WO2019006471A2 - Novel crispr rna targeting enzymes and systems and uses thereof - Google Patents

Novel crispr rna targeting enzymes and systems and uses thereof Download PDF

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WO2019006471A2
WO2019006471A2 PCT/US2018/040649 US2018040649W WO2019006471A2 WO 2019006471 A2 WO2019006471 A2 WO 2019006471A2 US 2018040649 W US2018040649 W US 2018040649W WO 2019006471 A2 WO2019006471 A2 WO 2019006471A2
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rna
crispr
protein
nucleic acid
cell
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WO2019006471A3 (en
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David R. Cheng
David A. Scott
Winston X. YAN
Shaorong Chong
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Arbor Biotechnologies Inc
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Arbor Biotechnologies Inc
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Priority to EP18765219.3A priority Critical patent/EP3645722A2/en
Priority to CA3068543A priority patent/CA3068543A1/en
Priority to US16/626,932 priority patent/US20210139890A1/en
Priority to AU2018291496A priority patent/AU2018291496B2/en
Publication of WO2019006471A2 publication Critical patent/WO2019006471A2/en
Publication of WO2019006471A3 publication Critical patent/WO2019006471A3/en
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Priority to AU2020201843A priority patent/AU2020201843B2/en
Priority to AU2021261919A priority patent/AU2021261919B2/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present disclosure relates to novel CRISPR systems and components, systems for detecting CRISPR systems, and methods and compositions for use of the CRISPR systems in, for example, nucleic acid targeting and manipulation.
  • CRISPR Clustered Regularly interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • the CRISPR-Cas systems of prokaryotic adaptive immunity are an extremely- diverse group of proteins effectors, non-coding elements, as well as loci architectures, some examples of which have been engineered and adapted to produce important biotechnologies.
  • the components of the systems involved in host defense include one or more effector proteins capable of modifying DNA or RNA and a RNA guide element that is responsible for targeting these protein activities to a specific sequence on the phage DNA or RNA.
  • the RN A guide is composed of a CRISPR RNA (crRNA) and may require an additional trans- activating RNA (tracrRNA) to enable targeted nucleic acid manipulation by the effector protein(s).
  • the crRNA consists of a direct repeat (DR) responsible for protein binding to the crRN A and a spacer sequence, which may be engineered to be complementary to a desired nucleic acid target sequence. In this way, CRISPR systems can be programmed to target DNA or RNA targets by modifying the spacer sequence of the crRNA.
  • DR direct repeat
  • CRISPR-Cas systems can be broadly classified into two classes: Class 1 systems are composed of multiple effector proteins that together form a complex around a crRNA, and Class 2 systems that consist of a single effector protein that complexes with the crRNA to target DNA or RNA substrates.
  • Class 1 systems are composed of multiple effector proteins that together form a complex around a crRNA
  • Class 2 systems that consist of a single effector protein that complexes with the crRNA to target DNA or RNA substrates.
  • the single-subunit effector compositions of the Class 2 systems provide a simpler component set for engineering and application translation, and has thus far been important sources of programmable effectors.
  • the discovery, engineering, and optimization of novel Class 2 systems may lead to widespread and powerful programmable technologies for genome engineering and beyond.
  • CRISPR-Cas systems are adaptive immune systems in archaea and bacteria that defend the species against foreign genetic elements.
  • the disclosure provides methods for computational identification of new single-effector CRISPR Class 2 sy stems from genomic databases, together with the development of the natural loci into engineered systems, and experimental validation and application translation.
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; and ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350), wherein the
  • RNA guide spacer sequence complementary to the RNA guide spacer sequence.
  • the effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the effector protein includes at least two HEPN domains. In some embodiments, none, one, or two or more of the HEPN domains are catalytically deactivated.
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR- associated protein or a nucleic acid encoding the CRISPR-associated protein; and iii) an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, wherein the WYL domain includes an amino acid sequence PXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid; and/or
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR- associated protein or a nucleic acid encoding the CRISPR-associated protein; and an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence
  • the CRISPR-associated protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
  • the activity is a nuclease activity (e.g., a DNAse activity, a targeted RNAse activity, or a collateral RNAse activity).
  • a nuclease activity e.g., a DNAse activity, a targeted RNAse activity, or a collateral RNAse activity.
  • the accessory protein increases the activity of the CRISPR-associated protein. In some embodiments, the accessory protein decreases the activity of the CRISPR-associated protein.
  • the accessory protein includes or consists of an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein includes or is RspWYLl (SEQ ID NO: 81).
  • the targeting of the target nucleic acid results in a modification of the target nucleic acid.
  • the CRISPR-associated protein is a Class 2 CRISPR-Cas system protein.
  • the CRISPR-associated protein includes a RuvC domain (e.g., at least one, two, three, or more RuvC domains).
  • the CRISPR-associated protein is selected from the group consisting of a Type VI Cas protein, a Type V Cas protein, and a Type II Cas protein.
  • the CRISPR- associated protein is a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, or a Cas9 protein.
  • the CRISPR-associated protein is a Type VI-D CRISPR-Cas effector protein comprising at least two HEPN domains, wherein none, one, or two or more of the HEPN domains are catalytically deactivated.
  • the effector protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein includes or is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the target nucleic acid is an RNA. In some embodiments, the target nucleic acid is a DNA.
  • the modification of the target nucleic acid is a cleavage event. In some embodiments, the modification results in: (a) decreased transcription; (b) decreased translation; or (c) both (a) and (b), of the target nucleic acid. In some embodiments, modification results in (a) increased transcription; (b) increased translation; or (c) both (a) and (b), of the target nucleic acid.
  • the effector protein includes one or more amino acid substitutions within at least one of the HEPN domains.
  • the one or more one amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • the one or more amino acid substitutions result in a reduction of an nuclease activity of the Type VI-D CRISPR-Cas effector protein, as compared to the nuclease activity of the Type VI-D CRISPR-Cas effector protein without the one or more acid substitutions.
  • the RNA guide includes a direct repeat sequence that includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs: 32-49, 52-77, 351-589).
  • the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G.
  • the direct repeat sequence includes or consists of either 5'-CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the spacer includes or consists of from about 15 to about 42 nucleotides.
  • the RNA guide further includes a trans-activating CRISPR RNA (tracrRNA).
  • the systems include a single-stranded donor template or a double-stranded donor template.
  • the donor template is a DNA or an RNA.
  • the systems include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
  • the systems are present in a delivery system (e.g., a nanoparticle, a liposome, an adeno-associated virus, an exosome, a microvesicle, and a gene- gun).
  • a delivery system e.g., a nanoparticle, a liposome, an adeno-associated virus, an exosome, a microvesicle, and a gene- gun.
  • the disclosure provides a cell including any of the systems described herein.
  • the cell is a eukaryotic cell (e.g., a mammalian cell or a plant cell).
  • the cell is a prokaryotic cell (e.g., a bacterial cell).
  • the disclosure provides an animal model or a plant model including a cell that includes any of the systems described herein.
  • the disclosure provides methods of cleaving a target nucleic acid (and compositions for use in such methods), which include contacting a target nucleic acid with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR-associated protein or the Type VI- D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR effector protein cleaves the target nucleic acid.
  • the target nucleic acid is within a cell.
  • the disclosure provides methods of inducing dormancy or death of a cell which include contacting the cell with a system described herein (and compositions for use in such methods), wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR-associated protein or the Type VI- D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell.
  • the death is via apoptosis, necrosis, necroptosis, or a combination thereof.
  • the target nucleic acid is an RNA selected from the group consisting of an mRNA, a tRNA, a ribosomal RNA, a non-coding RNA, a lncRNA, or a nuclear RNA.
  • the target nucleic acid is a DNA selected from the group consisting of chromosomal DNA, mitochondrial DNA, single- stranded DNA, or plasmid DNA.
  • the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein upon binding of the complex to the target nucleic acid, the CRISPR- associated protein or the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity.
  • the cell is a cancer cell (e.g., a tumor cell).
  • the cell is an infectious agent cell or a cell infected with an infectious agent.
  • the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
  • the disclosure provides methods of treating a condition or disease in a subject in need thereof and compositions for use in such methods.
  • the methods include administering to the subject a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject.
  • the condition or disease is a cancer or an infectious disease.
  • the condition or disease is cancer
  • the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Ho
  • the disclosure provides the use of a system described herein in a method selected from the group consisting of RNA sequence specific interference; RNA sequence-specific gene regulation; screening of RNA, RNA products, IncRNA, non-coding RNA, nuclear RNA, or mRNA; mutagenesis; inhibition of RNA splicing; fluorescence in situ hybridization; breeding; induction of cell dormancy; induction of cell cycle arrest; reduction of cell growth and/or cell proliferation; induction of cell anergy; induction of cell apoptosis; induction of cell necrosis; induction of cell death; or induction of programmed cell death.
  • the effector protein is fused to a base-editing domain, an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
  • the CRISPR-associated protein is fused to a base-editing domain (e.g., Adenosine Deaminase Acting on RNA (ADAR) 1 (ADAR1), ADAR2, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)), and activation-induced cytidine deaminase (AID)), an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
  • a base-editing domain e.g., Adenosine Deaminase Acting on RNA (ADAR) 1 (ADAR1), ADAR2, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)), and activation-induced cytidine deaminase (AID)
  • a base-editing domain e.g., Adenosine Deamin
  • the systems described herein include an RNA-binding fusion polypeptide that includes an RNA-binding domain (e.g., MS2) and a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID).
  • an RNA-binding domain e.g., MS2
  • a base-editing domain e.g., ADAR1, ADAR2, APOBEC, or AID.
  • the disclosure provides method of modifying an RNA molecule, comprising contacting the RNA molecule with a system described herein.
  • the disclosure provides methods of detecting a target RNA in a sample (and compositions for use in such methods).
  • the methods include: a) contacting the sample with: (i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex;
  • the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single- stranded target RNA in the sample.
  • the methods further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
  • the target RNA is single-stranded.
  • the target RNA is double-stranded.
  • the methods further include transcribing (e.g., using a T7 polymerase) a DNA molecule (e.g., a DNA molecule present in the sample) to produce the target RNA.
  • a DNA molecule e.g., a DNA molecule present in the sample
  • the target RNA was transcribed from a DNA molecule.
  • the methods further include pre-amplifying a nucleic acid in the sample (e.g., via isothermal amplification, recombinase polymerase amplification (RPA), or immunoprecipitation) prior to the contacting step.
  • RPA recombinase polymerase amplification
  • the methods further include contacting the sample with an accessory protein comprising at least one WYL domain.
  • the accessory protein includes an amino acid sequence having at least 80% (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6.
  • the accessory protein includes or is RspWYLl (SEQ ID NO: 81).
  • the effector protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1 -31 , and 200-350).
  • the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
  • the labeled detector RNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair.
  • the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.
  • a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
  • an amount of detectable signal produced by the labeled detector RNA is decreased. In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is increased.
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR-associated protein or a nucleic acid encoding the CRISPR-associated protein; and an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671); wherein the CRISPR-associated protein is capable of binding (e.g., bind
  • the accessory protein modulates an activity of the CRISPR-associated protein.
  • the activity is a nuclease activity (e.g., a DNAse activity or an RNAse activity).
  • the RNAse activity is targeted RNAse activity or a collateral RNAse activity.
  • the accessory protein increases the activity of the CRISPR-associated protein. In some embodiments, the accessory protein decreases the activity of the CRISPR-associated protein.
  • the accessory protein includes one WYL domain. In some embodiments, the accessory protein includes two WYL domains. In some embodiments, the accessory protein further includes a helix-tum-helix (HTH) fold. In some embodiments, the accessory protein further includes a ribbon-helix-helix (RHH) fold.
  • HTH helix-tum-helix
  • RHH ribbon-helix-helix
  • the accessory protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590- 671).
  • the accessory protein includes or consists of an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671).
  • the accessory protein is RspWYLl (SEQ ID NO: 81).
  • the target nucleic acid includes or is an RNA. In some embodiments, the target nucleic acid includes or is a DNA.
  • the targeting of the target nucleic acid results in a modification (e.g., a cleavage event) of the target nucleic acid.
  • the modification results in cell toxicity.
  • the modification results in decreased transcription and/or decreased translation of the target nucleic acid.
  • the modification results in increased transcription and/or increased translation of the target nucleic acid.
  • the CRISPR-associated protein is a Class 2 CRISPR-Cas system protein. In some embodiments, the CRISPR-associated protein includes a RuvC domain. In some embodiments, the CRISPR-associated protein is selected from the group consisting of a Type VI Cas protein, a Type V Cas protein, and a Type II Cas protein. In some embodiments, the CRISPR-associated protein is a Cas 13a protein, a Cas 13b protein, a Casl3c protein, a Casl2a protein, or a Cas9 protein.
  • the CRISPR-associated protein is a Type VI-D CRISPR-Cas effector protein comprising at least two HEPN domains (e.g., two, three, four, or more HEPN domains).
  • the Type VI-D CRISPR-Cas effector protein includes two HEPN domains.
  • at least one (e.g., one, two, three, four, or more) of the HEPN domains is catalytically inactivated.
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350).
  • the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the Type VI-D CRISPR-Cas effector protein includes or consists of one or more (e.g., two, three, four, five or six) amino acid substitutions within at least one of the HEPN domains. In some embodiments, the Type VI-D CRISPR-Cas effector protein includes six or less (e.g., five, four, three, two or one) amino acid substitutions within at least one of the HEPN domains. In some embodiments, the one or more one amino acid substitutions include or consist of an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • the one or more amino acid substitutions result in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more acid substitutions.
  • the CRISPR-associated proteins include or consist of at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the CRISPR-associated protein include or consist of at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and ⁇ is A or G.
  • the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G.
  • the direct repeat sequence includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589).
  • the direct repeat sequence includes or consists of either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the spacer includes from about 15 to about 42 nucleotides.
  • the RNA guide includes a trans-activating CRISPR RNA (tracrRNA).
  • the systems include a single- stranded donor template or a double-stranded donor template (e.g., a single-stranded DNA, a double stranded DNA, a single-stranded RNA, or a double stranded RNA).
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3 ' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X 3 is A or G, X 4 is C or G or T, X 5 is C or T, and Xe is A or G; and ii) a Type VI-D CRISPR- Cas effector protein or a nucleic acid encoding the effector protein
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X 4 is C or G or T, X 5 is C or T, and Xe is A or G; and ii) a Type VI-D CRISPR- Cas effector protein or a nucle
  • the Type VI-D CRISPR-Cas effector protein includes at least two HEPN domains.
  • the protein is about 1200 amino acids or less (e.g., 1100, 1000, 1050, 900, 950, 800 amino acids) in length.
  • the targeting of the target nucleic acid results in a modification of the target nucleic acid.
  • the modification of the target nucleic acid is a cleavage event.
  • the modification results in cell toxicity.
  • the modification results in decreased transcription and/or decreased translation of the target nucleic acid. In some embodiments, the modification results in increased transcription and/or increased translation of the target nucleic acid.
  • the systems further include a donor template nucleic acid.
  • the donor template nucleic acid is a DNA or an RNA.
  • the Type VI-D CRISPR-Cas effector protein includes one or more (e.g., two, three, four, five or six) amino acid substitutions within at least one of the HEPN domains.
  • the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • the one or more amino acid substitutions result in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more amino acid substitutions.
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350).
  • the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the systems include an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671).
  • the accessory protein includes two WYL domains.
  • the accessory protein further includes a helix-tum-helix (HTH) fold and/or a ribbon-helix-helix (RHH) fold.
  • the accessory protein is RspWYLl (SEQ ID NO: 81).
  • the accessory protein modulates (e.g., increases or decreases) an activity of the Type VI-D CRISPR-Cas effector protein.
  • the activity is an RNAse activity, an RNA-binding activity, or both.
  • the RNAse activity is a targeted RNAse activity or a collateral RNAse activity.
  • the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
  • the direct repeat sequence includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589). In some embodiments, the direct repeat sequence includes or consists of either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the spacer sequence includes or consists of from about 15 to about 42 nucleotides.
  • the systems provided herein include a single-stranded donor template or a double-stranded donor template (e.g., an RNA or a DNA molecule).
  • the systems provided herein include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein and/or a nucleic acid
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein and/or a
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein or a nucle
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of : i) an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; ii) a Type VI-D CRISPR-Cas effector protein
  • the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G; ii) a Type VI-D CRISPR-Ca
  • the accessory protein is RspWYLl (SEQ ID NO: 81).
  • the effector protein includes at least two HEPN domains. In some embodiments, the effector protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the CRISPR-associated protein (e.g., Type VI-D CRISPR-Cas effector protein) is fused to a base-editing domain (e.g., Adenosine Deaminase Acting on RNA (ADAR) 1; ADAR2; apolipoprotein B mRNA editing enzyme, catalytic polypepti de- like (APOBEC); and activation-induced cytidine deaminase (AID)).
  • ADAR Adenosine Deaminase Acting on RNA
  • ADAR2 apolipoprotein B mRNA editing enzyme, catalytic polypepti de- like
  • AID activation-induced cytidine deaminase
  • the base-editing domain is further fused to an RNA-binding domain.
  • the CRISPR associated protein (e.g., a Type VI-D CRISPR- Cas effector protein) is fused to a RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
  • the CRISPR-associated (e.g., a Type VI-D CRISPR-Cas effector protein) further includes a linker sequence.
  • the CRISPR- associated protein (e.g., a Type VI-D CRISPR-Cas effector protein) includes one or more mutations or amino acid substitutions that render the CRISPR-associated protein unable to cleave RNA.
  • the systems described herein also include an RNA-binding fusion polypeptide that includes an RNA-binding domain and a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, and AID).
  • a base-editing domain e.g., ADAR1, ADAR2, APOBEC, and AID.
  • the RNA-binding domain is MS2, PP7, or Qbeta.
  • the systems described herein include a nucleic acid encoding the CRISPR-associated protein (e.g., a Type VI-D CRISPR-Cas effector protein).
  • the nucleic acid encoding the CRISPR-associated protein is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
  • the nucleic acid encoding the CRISPR-associated protein is codon-optimized for expression in a cell (e.g., a mammalian cell or a bacterial cell).
  • the systems described herein include a nucleic acid encoding the accessory protein.
  • the nucleic acid encoding the accessory protein is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
  • the nucleic acid encoding the accessory protein is codon-optimized for expression in a cell.
  • the systems described herein include a nucleic acid encoding one or more RNA guides (e.g., crRNAs).
  • the nucleic acid encoding the one or more RNA guides is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
  • the systems described herein include a nucleic acid encoding a target nucleic acid (e.g., a target RNA).
  • a target nucleic acid e.g., a target RNA
  • the nucleic acid encoding the target nucleic acid is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
  • the systems described herein include a nucleic acid encoding a CRISPR-associated protein and a nucleic acid encoding an accessory protein in a vector.
  • the system further includes one or more nucleic acids encoding an RNA guide present in the vector.
  • the systems provided herein include a nucleic acid encoding a Type VI-D CRISPR-Cas effector protein in a vector.
  • the systems provided herein include a nucleic acid encoding the Type VI-D CRISPR-Cas effector protein and a nucleic acid encoding the accessory protein in a vector.
  • the system further includes one or more nucleic acids encoding one or more RNA guides (e.g., crRNAs) in the vector.
  • the vectors included in the systems are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors.
  • the vectors included in the system are phage vectors.
  • the systems provided herein are in a delivery system.
  • the delivery system is a nanoparticle, a liposome, an exosome, a microvesicle, and a gene-gun.
  • the disclosure also provides a cell (e.g., a eukaryotic cell or a prokaryotice cell (e.g., a bacterial cell)) comprising a system described herein.
  • the eukaryotic cell is a mammalian cell (e.g., a human cell) or a plant cell.
  • animal models e.g., rodent, rabbit, dog, monkey, or ape models
  • plant model that include the cells.
  • the disclosure provides methods of cleaving a target nucleic acid (and compositions for use in such methods), wherein the methods include contacting the target nucleic acid with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR- associated protein or the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR effector protein cleaves the target nucleic acid.
  • the methods include contacting the target nucleic acid with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR- associated protein or the Type VI-D CRISPR effector protein associate
  • the target nucleic acid is within a cell.
  • the disclosure also provides methods of inducing dormancy or death of a cell (and compositions for use in such methods), wherein the methods include contacting the cell with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell.
  • the death of the cell is via apoptosis, necrosis, necroptosis, or a combination thereof.
  • the target nucleic acid is an RNA molecule (e.g., an mRNA, a tRNA, a ribosomal RNA, a non-coding RNA, a IncRNA, or a nuclear RNA).
  • the target nucleic acid is a DNA molecule (e.g., chromosomal DNA, mitochondrial DNA, single-stranded DNA, or plasmid DNA).
  • the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein upon binding of the complex to the target nucleic acid, exhibits collateral RNAse activity.
  • the cell is a cancer cell (e.g., a tumor cell).
  • the cell is an infectious agent cell or a cell infected with an infectious agent.
  • the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
  • the disclosure provides methods of treating a condition or disease in a subject in need thereof (and compositions for use in such methods, the methods include administering to the subject a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject.
  • the condition or disease is a cancer or an infectious disease.
  • the condition or disease is cancer
  • the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer
  • the disclosure provides the use of a system described herein in a method selected from the group consisting of RNA sequence specific interference; RNA sequence-specific gene regulation; screening of RNA, RNA products, IncRNA, non-coding RNA, nuclear RNA, or mRNA; mutagenesis; inhibition of RNA splicing; fluorescence in situ hybridization; breeding; induction of cell dormancy; induction of cell cycle arrest; reduction of cell growth and/or cell proliferation; induction of cell anergy; induction of cell apoptosis; induction of cell necrosis; induction of cell death; or induction of programmed cell death.
  • the methods described herein are performed either in vitro, in vivo, or ex vivo.
  • the disclosure also provides methods of modifying an RNA molecule (and compostions for use in such methods), including contacting the RNA molecule with a system described herein.
  • the spacer sequence is complementary to at least 15 nucleotides of the RNA molecule.
  • the disclosure also provides methods of detecting a target RNA (e.g., a single- stranded RNA or a double-stranded RNA) in a sample, the methods including: a) contacting the sample with: (i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR- Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by
  • the Type VI-D CRISPR-Cas effector protein includes at least two HEPN domains. In some embodiments, the Type VI-D CRISPR-Cas effector protein is about 1200 amino acids or less in length.
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350).
  • the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
  • the effector protein includes one or more amino acid substitutions within at least one of the HEPN domains.
  • the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • the methods further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
  • the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
  • the labeled detector RNA includes a fluorescence-emitting dye pair. In some embodiments, the labeled detector RNA includes a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the labeled detector RNA includes a quencher/fluor pair.
  • FRET fluorescence resonance energy transfer
  • an amount of detectable signal produced by the labeled detector RNA is decreased. In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is increased. In some embodiments, the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.
  • a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
  • the labeled detector RNA includes a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof.
  • the disclosure relates to engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein XI is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and X6 is A or G; and a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is
  • the disclosure relates to engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5 '- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X 4 is C or G or T, X 5 is C or T, and Xe is A or G; and a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effect
  • the Type VI-D CRISPR-Cas effector proteins include at least two HEPN domains.
  • the Type VI-D CRISPR-Cas effector proteins include an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 1 , and SEQ ID NO: 10.
  • the Type VI-D CRISPR-Cas effector proteins include an amino acid sequence having at least 95% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1 -31 , and 200-350), or they can include an amino acid sequence provided in Table 2.
  • the direct repeat sequence can include a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589).
  • the targeting of the target nucleic acid results in a modification of the target nucleic acid.
  • the modification of the target nucleic acid can be a cleavage event.
  • the Type VI-D CRISPR-Cas effector proteins can include one or more amino acid substitutions within at least one of the HEPN domains resulting in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more amino acid substitutions, e.g., 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions.
  • the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • the Type VI-D CRISPR-Cas effector protein is fused to a base- editing domain, e.g., to an RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
  • the Type VI-D CRISPR-Cas effector protein includes at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or both.
  • the direct repeat sequence includes either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the spacer consists of from about 15 to about 42 nucleotides.
  • the systems include the nucleic acid encoding the Type VI-D CRISPR-Cas effector protein, operably linked to a promoter.
  • the promoter can be a constitutive promoter.
  • the nucleic acid encoding the Type VI-D CRISPR-Cas effector protein is codon-optimized for expression in a cell.
  • the nucleic acids encoding the Type VI-D CRISPR-Cas effector protein are operably linked to a promoter within in a vector, e.g., selected from the group consisting of a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector.
  • the system is present in a delivery system selected from the group consisting of a nanoparticle, a liposome, an exosome, a microvesicle, and a gene-gun.
  • the systems can further include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
  • the disclosure includes one or more cells that include the systems described herein.
  • the disclosure provides methods of cleaving a target nucleic acid.
  • the methods include contacting the target nucleic acid with a system as described herein; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid; wherein the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex;
  • the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence, the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid.
  • the disclosure provides methods of inducing dormancy or death of a cell, e.g., in vitro or in vivo (and compositions for use in such methods), the method including contacting the cell with a system as described herein; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid within the cell; wherein the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex; wherein the complex binds to the target nucleic acid sequence that is
  • the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell.
  • the cell can be a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
  • the disclosure provides methods of modifying a target nucleic acid in a sample, in which the methods include contacting the sample with a system as described herein, e.g., with fusion proteins; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid within the sample; wherein the Type VI-D CRISPR-Cas effector protein fused to the base editing domain associates with the RNA guide to form a complex; wherein the complex binds to the target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein after binding of the complex to the target nucleic acid sequence, the Type VI-D CRISPR-Cas effector protein fused to the base-editing domain modifies at least one nucleobase of the target nucleic acid.
  • the disclosure provides methods of detecting a single-stranded target RNA in a sample. These methods include: a) contacting the sample with: (i) a RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the label
  • the effector protein includes an amino acid sequence having at least 90% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1- 31, and 200-350). These methods can further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
  • cleavage event refers to a break in a target nucleic acid created by a nuclease (e.g., a Type VI-D CRISPR-Cas effector protein) of a CRISPR. system described herein.
  • the cleavage event is a single-stranded RNA break.
  • the cleavage event is a double-stranded RNA break.
  • the cleavage event is a double-stranded DNA break.
  • the cleavage event is a single-stranded DNA break.
  • CRISPR system or ' " Clustered Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) system” as used herein refer to nucleic acids and/or proteins involved in the expression of, or directing the activity of, CRISPR-efFectors, including sequences encoding CRISPR effectors, RNA guides, and other sequences and transcripts from a CRISPR locus.
  • CRISPR-efFectors including sequences encoding CRISPR effectors, RNA guides, and other sequences and transcripts from a CRISPR locus.
  • the CRISPR system is an engineered, non- naturally occurring CRISPR system
  • the components of a CRISPR system may include a nucleic acid(s) (e.g., a vector) encoding one or more components of the sy stem, a component(s) in protein form, or a combination thereof.
  • CRISPR array refers to the nucleic acid (e.g., DNA) segment that includes CRISPR repeats and spacers, starting with the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats.
  • CRISPR repeat/' or CRISPR direct repeat or “direct repeat,” as used herein, refer to multiple short direct repeating sequences, which show very little or no sequence variation within a CRISPR array.
  • CRISPR RNA or "crRNA” as used herein refers to a RNA molecule including a guide sequence used by a CRISPR effector to target a specific nucleic acid sequence.
  • crRNAs contains a sequence that mediates target recognition and a sequence that forms a duplex with a tracrRNA.
  • the crRNA: tracrRNA duplex binds to a CRISPR effector.
  • donor template or "donor template nucleic acid,” as used herein refers to a nucleic acid molecule that can be used by one or more cellular proteins to modify the sequence of a target nucleic acid after a CRISPR-associated protein described herein has altered the target nucleic acid.
  • the donor template nucleic acid is a double-stranded nucleic acid.
  • the donor template nucleic acid is a single-stranded nucleic acid.
  • the donor template nucleic acid is linear.
  • the donor template nucleic acid is circular (e.g., a plasraid).
  • the donor template nucleic acid is an exogenous nucleic acid molecule.
  • the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome).
  • the donor template is a DNA molecule.
  • the donor template is an RNA molecule.
  • CRISPR effector refers to a protein that carries out an enzymatic activity or that binds to a target site on a nucleic acid specified by a RNA guide.
  • a CRISP effector has endonuclease activity, nickase activity, exonuciease activity, transposase activity, and/or excision activity.
  • the CRISPR-associated protein is a Type VI Cas protein, a Type V Cas protein, or a Type II Cas protein.
  • the CRISPR-associated protein is a Casl 3a protein, a Cas 13b protein, a Casl3c protein, a Casl3d protein, a Casl2a protein, or a Cas9 protein.
  • the CRISPR-associated protein is a Type Vl-D CRISPR-Cas effector protein described herein.
  • RNA guide refers to any RNA molecule that facilitates the targeting of a protein described herein to a target nucleic acid.
  • RNA guides include, but are not limited to, crRNAs or crR As in combination with cognate trans- activating RNAs (tracrR As). The latter may be independent RNAs or fused as a single RNA using a linker.
  • the RNA guide is engineered to include a chemical or biochemical modification, in some embodiments, an RNA guide may include one or more nucleotides.
  • oil of replication refers to a nucleic acid sequence in a replicating nucleic acid molecule (e.g., a plasmid or a chromosome) that is recognized by a replication initiation factor or a DNA replicase.
  • a replicating nucleic acid molecule e.g., a plasmid or a chromosome
  • targeting refers to the ability of a complex including a CRISPR-associ ated protein and a RN A guide, such as a crRNA, to bind to a specific target nucleic acid and not to other nucleic acids that do not have the same sequence as the target nucleic acid.
  • target nucleic acid refers to a specific nucleic acid sequence that specifically binds to a complex including a CRlSPR-associated protein and a RNA guide described herein.
  • the target nucleic acid is or includes a gene.
  • the target nucleic acid is or includes a non-coding region (e.g. , a promoter).
  • the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.
  • trans-activating crRNA or "tracrRNA” as used herein refer to an RNA including a sequence that forms a structure required for a CRlSPR-associated protein to bind to a specified target nucleic acid.
  • RNAse activity refers to non-specific RNAse activity of a CRlSPR-associated protein after the enzyme has bound to and/or modified a specifically-targeted nucleic acid.
  • a CRlSPR-associated protem e.g., a Type VI-D CRISPR-Cas effector protem
  • exhibits collateral RNAse activity after binding to a target nucleic acid e.g., a target RNA
  • nucleic acid that is cleaved or degraded by a CRlSPR-associated protein in a non-specific manner is referred to herein as a "non-target nucleic acid.”
  • FIG. 1 depicts a schematic representation of a maximum likelihood tree topology for an exemplary subset of Casl3d, with the genomic arrangement of the genes encoding predicted protein components of Type VI-D system components shown to the right. Each locus sequence is identified by a protein accession or gene number, with the species name provided where available. Key proteins and CRISPR arrays are depicted as follows: white - Casl3d, horizontal stripes - WYL1 accessory protein, light gray - WYL domain containing protein, vertical stripes - Casl, dark gray - Cas2.
  • FIG. 2A depicts a schematic tree comparing the different type VI subtype locus structures. Gene arrows are shown roughly proportional to size. Labels denote the following: WYL - WYL domain, HEPN - HEPN nuclease domain.
  • FIG. IB depicts a size comparison for Casl3 proteins from the 4 type VI subtypes; error bars specify the mean and standard deviation.
  • FIG. 3 depicts a phylogenetic tree of Casl proteins from type II and type VI CRISPR- Cas systems. The tree was constructed for a non-redundant set of Casl proteins associated with Casl 3d and type II and type VI CRISPR-Cas systems as described previously (see (Peters et al, 2017)). Several Casl proteins associated with subtype I-E systems were selected for an outgroup. Each sequence is denoted by a local numeric identifier, CRISPR- Cas type and species name (if available). Casl proteins associated with Casl3d are denoted by "CAS-VI-D", and those associated with Casl3a by "CAS-VI-A". Several branches were collapsed and are shown by triangles with CRISPR-Cas system indicated on the right.
  • FIGs. 4A and 4B depict a phylogenetic tree constructed for a combined set of Casl3d sequences described (light gray) and previously described Casl3a sequences. Each sequence is denoted by a protein locus tag and species name (if available). Casl3d proteins form a clade with a 100% bootstrap support value (shown on branch).
  • FIGs. 5A, 5B and5C depict a multiple sequence alignment of Casl3d protein sequences (RspCasl3d (SEQ ID NO: 2) and EsCasl3d (SEQ ID NO: 1) and Casl3a protein sequences (LbaCasl3a (SEQ ID NO: 156), LbuCasl3a (SEQ ID NO: 157), LshCasl3a (SEQ ID NO: 158)).
  • Previously identified domains of Cas 13a are highlighted with varying background shading as indicated in the figure (NTD, N-terminal domain). Note the nearly complete absence of a counterpart to the Helical-1 domain of Casl3a in Casl3d (the alignment in this region cannot be considered reliable).
  • FIG. 6 depicts a phylogenetic tree of the WYLl protein family.
  • Exemplary WYLl proteins associated with Casl3d are denoted by gray.
  • CRISPR CRISPR
  • Several branches were collapsed and are indicated by triangles. Domain organization is schematically shown next to each branch. Abbreviation: WYL - WYL domain (usually fused to a characteristic C-terminal
  • RHH - ribbon helix helix superfamily DNA binding domain RHH - ribbon helix helix superfamily DNA binding domain
  • FIG. 7 depicts a multiple sequence alignment of exemplary WYLl protein sequences.
  • the RHH domain is denoted by 'r' and the WYL domain fused to the characteristic C- terminal subdomain is denoted by 'y' underneath the alignment.
  • the predicted secondary structure elements are shown (E, extended conformation ( ⁇ -strand), H, oc-helix).
  • FIG. 8 depicts a design of minimal engineered CRISPR-Cas systems for the Rsp and Es type VI-D CRISPR loci (referred to as RspCasl3d and EsCasl3d systems), with a spacer library tiling pACYC184 (both top strand and bottom strand).
  • FIG. 9 depicts a schematic of the bacterial negative selection screen used to evaluate functional parameters of RspCasl3d and EsCasl3d systems.
  • FIGs. 10A and 10B depict a negative control condition from bacterial screens for EsCasl3d and RspCasl3d systems, respectively. Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library.
  • Non-targeting CRISPR arrays (with spacers matching a GFP open reading frame) inserted into EsCasl3d and RspCasl3d screening systems showed minimal levels of depletion in bacterial negative selection screens (no GFP open reading frame was included in our screen system).
  • FIGs. 11A and 11B depict a negative control condition from bacterial screens for EsCasl3d and RspCasl3d systems, respectively. Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library. Deletion of EsCasl3d and RspCasl3d-RspWYLl open reading frames from the EsCasl3d and
  • FIGs. 12A and 12B depict the distribution and magnitude of crRNA depletion from bacterial screens for EsCasl3d and RspCasl3d, respectively.
  • Depletion value was calculated by normalized sequencing reads from the screen output divided by normalized reads from the pre-transformation screen input library for each crRNA spacer and orientation.
  • Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library, cloned into the screening library.
  • the vertical dashed lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
  • FIGs. 13A and 13B depict the location of strongly depleted targets of the active DR orientation over the strands and genetic features of the pACYC184 plasmid for EsCasl3d and RspCasl3d systems, respectively.
  • Light gray outlines represent the total number of spacers (y-axis) targeting a location, while short bars depict the locations of strongly depleted spacers with heatmap color proportional to magnitude of depletion.
  • Directional expression data for pACYC184 is plotted as a heatmap between the x-axes.
  • FIGs 14A and 14B depict web logos for the 5' and 3' 30 nt regions flanking strongly depleted targets for EsCasl3d and RspCasl3d systems, and show no evidence of PFS or PAM requirements.
  • FIG. 14C depicts violin plots of bit scores of all possible PFS targeting rules of up to length 3 involving the target site and +/- 15 nt flanking region, for BzCasl3b, RspCasl3d, and EsCasl3d systems. Dots represent data points outside of the discernable density of the violin plot. These dots accurately recapitulate the known PFS positions of BzCasl3b, as shown above the dots.
  • FIG. 15 depicts bar charts showing the fraction of hits for RspCasl3d and EsCasl3d systems according to features of the plasmid for all targets.
  • FIGs. 16A and 16B depict heatmaps of the fraction (# strongly depleted spacers) / (# strongly depleted spacers + # non-depleted spacers) for all target regions (CRISPR arrays with active direct repeat orientation only) with no predicted secondary structure between specific start (x-axis) and end (y-axis) locations.
  • White boxes indicate specific target regions (bounded by start (x-axis) and end (y-axis) locations), where selection of spacers with no predicted secondary structure maximized targeting efficacy, while minimizing the number of screen spacers eliminated due to the presence of predicted secondary structure.
  • Targets these spacer populations are referred to as "low secondary structure targets" for RspCasl3d and EsCasl3d respectively.
  • FIG. 16C depicts bar charts showing the fraction of hits for RspCasl3d
  • EsCasl3d systems according to features of the plasmid for low secondary structure targets.
  • FIG. 17 depicts a schematic of the RNA extraction from bacterial screen, next- generation sequencing (NGS), and alignment to determine the mature crRNA for EsCasl3d. Distribution of read counts by crRNA sequence location is depicted on the right, and the predicted EsCasl3d mature crRNA secondary structure is shown.
  • NGS next- generation sequencing
  • FIG. 18 depicts a coomassie blue stained polyacrylamide gel of purified recombinant proteins EsCasl3d, RspCasl3d, and RspWYLl respectively.
  • FIG. 19 depicts schematic representions of the major products identified from next- generation sequencing of in vitro cleaved RNA fragments from the pre-crRNA processing with EsCasl3d and RspCasl3d.
  • the black line represents the direct repeats and associated secondary structure
  • the box represents the full-length spacer
  • the filled triangle represents the cleavage sites.
  • the lengths described are for processed EsCasl3d crRNAs, with RspCasl3d having one extra nucleotide due to the 31nt natural length spacer used for instead of 30. Not depicted are the 3-4 nt at the 5' end of the pre-crRNA from T7 in vitro transcription.
  • FIGs. 20A, 20B, 20C, and 20D depict denaturing gels displaying Casl3d mediated cleavage of their cognate pre-crRNAs over a dose titration of effector concentration.
  • the dependence of Casl3d crRNA biogenesis on divalent metal cations was evaluated with the introduction of lOOmM EDTA to the standard reaction conditions.
  • FIG. 21 depicts a denaturing gel displaying LwaCasl3a at a final concentration of lOOnM processing of pre-crRNA (200nM) without the presence of EDTA, and under reaction conditions supplemented with increasing concentrations of EDTA (3.3 - lOOmM).
  • FIGs. 22A and 22B depict a titration of Apo EsCasl3d and RspCasl3d (100 - 0.4nM) over a non-targeted ssDNA substrate ( ⁇ ).
  • FIGs. 23A and 23B depict a titration of EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over non-targeted ssDNA substrates ( ⁇ ).
  • FIGs. 24A and 24B depict a titration of EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over targeted ssDNA substrates ( ⁇ ). Saturation of target cleavage activity was observed at approx. 50nM RspCasl3d-crRNA complex and ⁇ EsCasl3d- crRNA complex.
  • FIGs. 25A and 25B depict representative denaturing gels displaying the targeted RNase activity of EsCasl3d and RspCasl3d effector proteins, with substrate RNA cleavage occurring when the crRNA matches its complementary target ssRNA.
  • RNA substrates are 5' labeled with IRDye 800.
  • FIGs. 26A and 26B depict representative denaturing gels displaying non-specific RNase activity of the Casl3d effectors upon targeted substrate recognition, demonstrated by the cleavage of fluorescein dUTP body -labeled collateral RNA upon activation of the target nuclease activity.
  • EsCasl3d-crRNA and RspCasl3d-crRNA complexes were formed by pre-incubating Casl3d and cognate crRNA for 5 minutes at 37°C, prior to adding target and/or collateral ssRNA and incubating the reaction for 30 minutes.
  • FIGs. 26C and 26D depict denaturing gels displaying cleavage reactions of the Casl3d-crRNA complex over two distinct ssRNA substrates, short 150nt target RNAs (top) and longer 800nt fluorescent body-labeled ssRNA substrates (bottom) for EsCasl3d and RspCasl3d.
  • the labels A and B correspond to matching crRNA / substrate pairs.
  • FIG. 27A depicts a comparative depletion plot of bacterial screens performed on RspCasl3d only (solid line, long dashes) versus RspCasl3d with RspWYLl (short and medium dashes).
  • the dashed vertical lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
  • FIG. 27B depicts spacer depletion ratios for RspCasl3d with and without RspWYLl.
  • FIG. 28 depicts a depletion plot of bacterial screens using only RspWYLl and the repeat-spacer-repeat library associated with RspCasl3d.
  • FIG. 29A and 29B depict representative activity of titrating different molar ratios of purified RspWYLl to a fixed dose of RspCasl3d.
  • FIG. 29A is an ssRNA substrate cleavage assay, and FIG. 29B evaluate the effect of RspWYLl on collateral activity.
  • FIG. 29C depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of Apo RspCasl3d (200nM) for target ssRNA.
  • FIG. 29D depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of Apo RspCasl3d (200nM) for collateral ssRNA activity.
  • FIG. 29E depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of RspCasl3d-crRNA complex (50nM) for target ssRNA.
  • FIG. 29F depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of RspCasl3d-crRNA complex (50nM) for collateral ssRNA activity.
  • FIGs. 30A and 30B depict representative activity of titrating different molar ratios of purified RspWYLl to a fixed dose of EsCasl3d.
  • FIG. 30A is an ssRNA substrate cleavage assay, and FIG. 30B evaluate the effect of RspWYLl on collateral activity of EsCasl3d.
  • RspWYLl was pre-incubated along with the pre-crRNA and Casl3d effector for 5 minutes at 37°C before incubation with substrate RNA.
  • the final concentration of Casl3d in the reaction is 33nM with a 2: 1 ratio of Casl3d to pre-crRNA.
  • FIG. 31 shows that RspWYLl enhances the activity of type VI-B effector BzCasl3b.
  • Representative gel displaying the ability of RspWYLl to enhance target cleavage and collateral activity for Casl3 enzymes of subtype VI-B, demonstrating modularity beyond Type VI-D.
  • RspWYLl was pre-incubated along with the pre-crRNA and BzCasl3b effector for 5 minutes at 37C before incubation with substrate RNA.
  • FIGs. 32A and 32B show that EsCasl3d and RspCasl Sd, respectively, are capable of specific detection of RNA species using the collateral effect of the enzymes, and additionally, demonstrate differential activity over short ribonucleotide oligomer substrates.
  • the poly-G and poly-U labels refer to substrates containing 5 identical ribonucleotide bases, with the 5' end modified with a FAM labeled fluorescent nbonucleotide and the 3' end modified with an Iowa Black FQ fluorescent quencher. These data were collected 60 minutes after incubation at 37 C C. The error bars represent S.E.M. of four technical replicates.
  • FIGs. 33A and 333B depict the distribution and magnitude of crRNA depletion for primary screening of EsCasl3d and RspCasl 3d (effector only), respectively, in the absence of tetracycline.
  • the value of crRNA depletion was calculated by normalized sequencing reads from the screen output divided by normalized reads from the pre-transformation screen input library for each crRNA spacer and orientation.
  • the vertical dashed lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
  • FIGs. 34A and 34B depict the location of strongly depleted targets of the active DR orientation over the strands and genetic features of the pACYC i84 plasnnd for EsCasBd and RspCaslSd (effector only), respectively.
  • Light gray outlines represent the total number of spacers (y-axis) targeting a location, while short horizontal bars depict the locations of strongly depleted spacers with heatmap color proportional to magnitude of depletion.
  • CRISPR Class 2 effectors having two strictly conserved RX4-6H motifs, characteristic of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains.
  • CRISPR Class 2 effectors that contain two HEPN domains have been previously characterized and include, for example, CRISPR Casl3a (C2c2), Casl3b, and Casl3c.
  • HEPN domains have been shown to be RNAse domains and confer the ability bind to and cleave any target RNA molecule.
  • a HEPN domain comprises the amino acid sequence RXXXXH, wherein X is any amino acid (SEQ ID NO: 94).
  • the target RNA may be any suitable form of RNA, including but not limited to mRNA, tRNA, ribosomal RNA, non-coding RNA, lincRNA, and nuclear RNA.
  • the CRISPR-associated protein recognizes and cleaves targets located on the coding strand of open reading frames (ORFs).
  • the disclosure provides a family of CRISPR Class 2 effectors, referred to herein generally as Type VI-D CRISPR-Cas effector proteins, Casl3d or CasBe.
  • Type VI-D CRISPR-Cas effector proteins are significantly smaller (e.g., 20% fewer amino acids), and have less than 10% sequence similarity in multiple sequence alignments to other previously described effector proteins.
  • CRISPR Class 2 effectors can be used in a variety of applications, and are particularly suitable for therapeutic applications since they are significantly smaller than other effectors (e.g., CRISPR Casl3a, Casl3b, or Casl3c effectors) which allows for the packaging of the effectors and/or nucleic acids encoding the effectors into delivery systems having size limitations.
  • CRISPR Casl3a, Casl3b, or Casl3c effectors e.g., CRISPR Casl3a, Casl3b, or Casl3c effectors
  • Type VI-D CRISPR-Cas systems include a single effector
  • the CRISPR array includes direct repeat sequences typically 36 nucleotides in length, which are generally well conserved, especially on the 3' end which ends with TNTNAAAC (SEQ ID NO: 154). Reduced consensus of the nucleotide sequence in the 5' end of the direct repeats suggests that the crRNA is processed from the 5' end. With few exceptions, the 21 nucleotide sequence immediately upstream of the 3' end TNTNAAAC (SEQ ID NO: 154) starts with a highly conserved A and exhibits sequence complementarity that suggests strong base pairing for an RNA loop structure.
  • the spacers contained in the Casl3d CRISPR arrays are most commonly 30 nucleotides in length, with the majority of variation in length contained in the range of 28 to 36 nucleotides.
  • Type VI-D CRISPR-Cas effector proteins are provided below in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • a Type VI-D CRISPR-Cas effector proteins include an amino acid sequence having at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of any one of Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • a Type VI-D CRISPR-Cas effector proteins includes the amino acid sequence of any one of Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350).
  • the Type VI-D CRISPR-Cas effector proteins is DS499551 (SEQ ID NO: 1 ; also referred to herein as EsCasl3d) or LARF01000048 (SEQ ID NO: 2; also referred to herein as RspCasl3d), the amino acid sequences of each are provided below:
  • the CRISPR-associated proteins described herein are from about 800 to about 1150 amino acids long, such as about 850 to about 1100 amino acids in length, e.g., about 850 to about 1050, about 850 to about 1000 amino acids long, or about 850 to about 950 amino acids long.
  • the CRISPR-associated proteins e.g., Type VI-D CRISPR- Cas effector proteins
  • RNAse activity e.g., collateral RNAse activity
  • the CRISPR-associated proteins have DNAse activity.
  • the DNAse and/or RNAse activity is mediated by a single or both HEPN domains present in the CRISPR-associated proteins.
  • a CRISPR-associated protein (e.g., Type VI-D CRISPR-Cas effector protein) is derived from a Ruminococcus or Eubacterium bacterium. In some embodiments, the CRISPR associated protein is derived from a human stool sample bacterial source.
  • a complex comprised of (but not limited to) a CRISPR- associated protein and a crRNA is activated upon binding to a target nucleic acid (e.g., a target RNA). Activation induces a conformational change, which results in the complex acting as a non-specific RNase, cleaving and/or degrading nearby RNA molecules (e.g., ssRNA or dsRNA molecules) (i.e. , "collateral" effects).
  • a target nucleic acid e.g., a target RNA
  • Activation induces a conformational change, which results in the complex acting as a non-specific RNase, cleaving and/or degrading nearby RNA molecules (e.g., ssRNA or dsRNA molecules) (i.e. , "collateral" effects).
  • a complex comprised of (but not limited to) the CRISPR- associated protein and a crRNA does not exhibit collateral RNase activity subsequent to target recognition.
  • This "collateral-free” embodiment may comprise wild-type or engineered effector proteins.
  • a CRISPR-associated protein e.g., a Type VI-D CRISPR-Cas effector protein described herein
  • a CRISPR-associated protein recognizes and cleaves the target nucleic acid without any additional requirements adjacent to or flanking the protospacer (i.e., protospacer adjacent motif "PAM” or protospacer flanking sequence "PFS" requirements).
  • PAM protospacer adjacent motif
  • PFS protospacer flanking sequence
  • the CRISPR-associated proteins described herein have nuclease activity
  • the CRISPR-associated proteins can be modified to have diminished nuclease activity, e.g., nuclease inactivation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type CRISPR-associated proteins.
  • the nuclease activity can be diminished by several methods known in the art, e.g., introducing mutations into the nuclease domains of the proteins.
  • catalytic residues for the nuclease activities are identified, and these amino acid residues can be substituted by different amino acid residues (e.g., glycine or alanine) to diminish the nuclease activity.
  • the amino acid substitution is a conservative amino acid substitution.
  • the amino acid substitution is a non-conservative amino acid substitution.
  • the CRISPR-associated proteins described herein are modified to comprise one or more mutations (e.g., amino acid deletions, insertions, or substitutions) in at least one HEPN domain.
  • the CRISPR associate protein includes one, two, three, four, five, six, seven, eight, nine, or more amino acid substitutions in at least one HEPN domain.
  • the one or more mutations comprise asubstitution (e.g., an alanine substitution) at an amino acid residue corresponding to R295, H300, R849, H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
  • a substitution e.g., an alanine substitution
  • the presence of at least one of these mutations results in a CRISPR-associated protein having reduced nuclease activity (e.g., RNAse activity) as compared to the nuclease activity of the CRISPR-associated protein from which the protein was derived (i.e., lacking the mutation).
  • the inactivated CRISPR-associated proteins can be fused or associated with one or more functional domains (e.g., via fusion protein, linker peptides, "GS" linkers, etc.). These functional domains can have various activities, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, base-editing activity, and switch activity (e.g., light inducible).
  • methylase activity e.g., demethylase activity
  • transcription activation activity e.g., transcription repression activity
  • transcription release factor activity e.g., histone modification activity
  • RNA cleavage activity e.g., DNA cleavage activity
  • nucleic acid binding activity e.g., base-editing activity
  • switch activity e.g., light inducible
  • the functional domains are Kruppel associated box (KRAB), VP64, VP16, Fokl, P65, HSF1, MyoDl, Adenosine Deaminase Acting on RNA (ADAR) 1, ADAR2, APOBEC, cytidine deaminase (AID), mini-SOG, APEX, and biotin-APEX.
  • the functional domain is a base editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID).
  • the CRISPR-associated protein is fused to one functional domain.
  • the CRISPR-associated protein is fused to multiple (e.g., two, three, four, five, six, seven, eight, or more) functional domains.
  • the functional domain e.g., a base editing domain
  • an RNA- binding domain e.g., MS2
  • the CRISPR-associated protein is associated to or fused to a functional domain via a linker sequence (e.g., a flexible linker sequence or a rigid linker sequence). Exemplary linker sequences and functional domain sequences are provided in Table 10.
  • the positioning of the one or more functional domains on the inactivated CRISPR- associated proteins is one that allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect.
  • the functional domain is a transcription activator (e.g., VP16, VP64, or p65)
  • the transcription activator is placed in a spatial orientation that allows it to affect the transcription of the target.
  • a transcription repressor is positioned to affect the transcription of the target
  • a nuclease e.g., Fokl
  • the functional domain is positioned at the N-terminus of the CRISPR-associated protein.
  • the functional domain is positioned at the C-terminus of the CRISPR- associated protein.
  • the inactivated CRISPR-associated protein is modified to comprise a first functional domain at the N-terminus and a second functional domain at the C-terminus.
  • the present disclosure also provides a split version of the CRISPR-associated proteins described herein (e.g., a Type VI-D CRISPR-Cas effector protein).
  • the split version of the CRISPR-associated protein may be advantageous for delivery.
  • the CRISPR-associated proteins are split into two parts of the enzyme, which together substantially comprise a functioning CRISPR-associated protein.
  • the split can be done in a way that the catalytic domain(s) are unaffected.
  • the CRISPR-associated protein may function as a nuclease or may be an inactivated enzyme, which is essentially a RNA-binding protein with very little or no catalytic activity (e.g., due to mutation(s) in its catalytic domains).
  • Split enzymes are described, e.g., in Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat 'l. Acad. Sci. , 112.10 (2015): 2984-2989, which is incorporated herein by reference in its entirety.
  • the nuclease lobe and a-helical lobe are expressed as separate polypeptides.
  • the crRNA recruits them into a ternary complex that recapitulates the activity of full-length CRISPR-associated proteins and catalyzes site-specific DNA cleavage.
  • the use of a modified crRNA abrogates split-enzyme activity by preventing dimerization, allowing for the development of an inducible dimerization system.
  • the split CRISPR-associated protein can be fused to a dimerization partner, e.g., by employing rapamycin sensitive dimerization domains. This allows the generation of a chemically inducible CRISPR-associated protein for temporal control of the activity of the protein.
  • the CRISPR-associated protein can thus be rendered chemically inducible by being split into two fragments and rapamycin-sensitive dimerization domains can be used for controlled re-assembly of the protein.
  • the split point is typically designed in silico and cloned into the constructs. During this process, mutations can be introduced to the split CRISPR-associated protein and nonfunctional domains can be removed.
  • the two parts or fragments of the split CRISPR-associated protein i.e., the N-terminal and C-terminal fragments
  • the CRISPR-associated proteins described herein can be designed to be self-activating or self-inactivating.
  • the target sequence can be introduced into the coding construct of the CRISPR-associated protein.
  • the CRISPR-associated protein can cleave the target sequence, as well as the construct encoding the protein thereby self-inactivating their expression.
  • Methods of constructing a self-inactivating CRISPR system are described, e.g., in Epstein, and Schaffer, Mol. Ther. 24 (2016): S50, which is incorporated herein by reference in its entirety.
  • an additional crRNA expressed under the control of a weak promoter (e.g., 7SK promoter), can target the nucleic acid sequence encoding the CRISPR-associated protein to prevent and/or block its expression (e.g., by preventing the transcription and/or translation of the nucleic acid).
  • the transfection of cells with vectors expressing the CRISPR-associated protein, the crRNAs, and crRNAs that target the nucleic acid encoding the CRISPR-associated protein can lead to efficient disruption of the nucleic acid encoding the CRISPR-associated protein and decrease the levels of CRISPR-associated protein, thereby limiting the genome editing activity.
  • the genome editing activity of the CRISPR-associated protein can be modulated through endogenous RNA signatures (e.g., miRNA) in mammalian cells.
  • a CRISPR-associated protein switch can be made by using a miRNA-complementary sequence in the 5'-UTR of mRNA encoding the CRISPR-associated protein.
  • the switches selectively and efficiently respond to miRNA in the target cells.
  • the switches can differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Therefore, the switch systems can provide a framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information (see, e.g., Hirosawa et al. Nucl. Acids Res., 2017, 45(13): el 18).
  • the CRISPR-associated proteins can be inducibly expressed, e.g., their expression can be light-induced or chemically-induced. This mechanism allows for activation of the functional domain in the CRISPR-associated proteins. Light inducibility can be achieved by various methods known in the art, e.g., by designing a fusion complex wherein CRY2PHR/CIBN pairing is used in split CRISPR- associated proteins (see, e.g., Konermann et al. "Optical control of mammalian endogenous transcription and epigenetic states," Nature, 500.7463 (2013): 472).
  • Chemical inducibility can be achieved, e.g., by designing a fusion complex wherein FKBP/FRB (FK506 binding protein / FKBP rapamycin binding domain) pairing is used in split CRISPR-associated proteins. Rapamycin is required for forming the fusion complex, thereby activating the CRISPR-associated proteins (see, e.g., Zetsche, Volz, and Zhang, "A split-Cas9 architecture for inducible genome editing and transcription modulation," Nature Biotech., 33.2 (2015): 139-142).
  • FKBP/FRB FK506 binding protein / FKBP rapamycin binding domain
  • expression of the CRISPR-associated proteins can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system), hormone inducible gene expression system (e.g., an ecdysone inducible gene expression system), and an arabinose-inducible gene expression system.
  • inducible promoters e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system), hormone inducible gene expression system (e.g., an ecdysone inducible gene expression system), and an arabinose-inducible gene expression system.
  • expression of the RNA targeting effector protein can be modulated via a riboswitch, which can sense a small molecule like tetracycline (see, e.g., Goldfiess, Stephen J. et al. "Direct and specific chemical control of
  • inducible CRISPR-associated proteins and inducible CRISPR systems are described, e.g., in US Patent No. 8,871,445, US Publication No.
  • the CRISPR-associated proteins include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Localization Signal (NLS) attached to the N-terminal or C- terminal of the protein.
  • NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence
  • PKKKRKV (SEQ ID NO: 135); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 136)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 137) or
  • RQRRNELKRSP (SEQ ID NO: 138); the hRNPAl M9 NLS having the sequence
  • NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY SEQ ID NO: 139
  • sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV SEQ ID NO: 140
  • VSRKRPRP SEQ ID NO: 141
  • PPKKARED SEQ ID NO: 142
  • PQPKKKPL SEQ ID NO: 143
  • SALIKKKKKMAP SEQ ID NO: 144 of mouse c-abl IV
  • the sequences DRLRR SEQ ID NO: 145) and PKQKKRK(SEQ ID NO: Hepatitis virus delta antigen
  • sequence REKKKFLKRR SEQ ID NO: 148) of the mouse Mxl protein
  • sequence KRKGDEVDGVDEVAKKKSKK SEQ ID NO: 149) of the human poly(ADP-ribo
  • the CRISPR- associated protein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Export Signal (NES) attached the N-terminal or C-terminal of the protein.
  • NES Nuclear Export Signal
  • a C-terminal and/or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.
  • the CRISPR-associated proteins described herein are mutated at one or more amino acid residues to alter one or more functional activities.
  • the CRISPR-associated protein is mutated at one or more amino acid residues to alter its helicase activity.
  • the CRISPR-associated protein is mutated at one or more amino acid residues to alter its nuclease activity (e.g., endonuclease activity or exonuclease activity).
  • the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with an RNA guide.
  • the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid.
  • the CRISPR-associated proteins described herein are capable of cleaving a target nucleic acid molecule.
  • the CRISPR-associated protein cleaves both strands of the target nucleic acid molecule.
  • the CRISPR-associated protein is mutated at one or more amino acid residues to alter its cleaving activity.
  • the CRISPR-associated protein may comprise one or more mutations that render the enzyme incapable of cleaving a target nucleic acid.
  • the CRISPR-associated protein comprise one or more mutations such that the enzyme is capable of cleaving a single strand of the target nucleic acid (i.e. , nickase activity).
  • the CRISPR-associated protein is capable of cleaving the strand of the target nucleic acid that is complementary to the strand to which the RNA guide hybridizes. In some embodiments, the CRISPR-associated protein is capable of cleaving the strand of the target nucleic acid to which the guide RNA hybridizes.
  • a CRISPR-associated protein described herein can be engineered to have a deletion in one or more amino acid residues to reduce the size of the enzyme while retaining one or more desired functional activities (e.g., nuclease activity and the ability to interact functionally with a RNA guide).
  • the truncated CRISPR-associated protein can be advantageously used in combination with delivery systems having load limitations.
  • nucleic acids encoding the proteins e.g., a CRISPR-associated protein or an accessory protein
  • RNA guides e.g., a crRNA
  • the nucleic acid is a synthetic nucleic acid.
  • the nucleic acid is a DNA molecule.
  • the nucleic acid is an RNA molecule (e.g., an mRNA molecule).
  • the nucleic acid is an mRNA.
  • the mRNA is capped, polyadenylated, substituted with 5-methylcytidine, substituted with pseudouridine, or a combination thereof.
  • the nucleic acid e.g., DNA
  • a regulatory element e.g., a promoter
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a cell-specific promoter.
  • the promoter is an organism-specific promoter.
  • Suitable promoters include, for example, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a HI promoter, retroviral Rous sarcoma virus LTR promoter, a
  • cytomegalovirus (CMV) promoter a SV40 promoter, a dihydrofolate reductase promoter, and a ⁇ -actin promoter.
  • CMV cytomegalovirus
  • SV40 promoter a SV40 promoter
  • dihydrofolate reductase promoter a SV40 promoter
  • ⁇ -actin promoter a ⁇ -actin promoter.
  • U6 promoter can be used to regulate the expression of an RNA guide molecule described herein.
  • the nucleic acid(s) are present in a vector (e.g., a viral vector or a phage).
  • the vectors can include one or more regulatory elements that allow for the propagation of the vector in a cell of interest (e.g., a bacterial cell or a mammalian cell).
  • the vector includes a nucleic acid encoding a single component of a CRISPR-associated (Cas) system described herein.
  • the vector includes multiple nucleic acids, each encoding a component of a CRISPR-associated (Cas) system described herein.
  • the present disclosure provides nucleic acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequences described herein.
  • the present disclosure also provides amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences described herein.
  • the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100
  • nucleotides e.g., contiguous or non-contiguous nucleotides
  • the nucleic acid sequences have at least a portion (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from the sequences described herein.
  • the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as the sequences described herein.
  • the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from the sequences described herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes should be at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100% of the length of the reference sequence.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the CRISPR-associated proteins and accessory proteins described herein can be fused to one or more peptide tags, including a His-tag, GST-tag, or myc-tag.
  • the CRISPR-associated proteins or accessory proteins described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein or yellow fluorescent protein).
  • the proteins described herein can be delivered or used as either nucleic acid molecules or polypeptides.
  • the nucleic acid molecule encoding the CRISPR-associated proteins can be codon-optimized.
  • the nucleic acid can be codon optimized for use in any organism of interest, in particular human cells or bacteria.
  • the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA).
  • the CRISPR systems described herein include at least RNA guide (e.g., a crRNA).
  • RNA guide e.g., a crRNA
  • the architecture of multiple RNA guides is known in the art (see, e.g., International Publication Nos. WO 2014/093622 and WO 2015/070083, the entire contents of each of which are incorporated herein by reference).
  • the CRISPR systems described herein include multiple RNA guides (e.g., one, two, three, four, five, six, seven, eight, or more RNA guides).
  • the RNA guide includes a crRNA.
  • the RNA guide includes a crRNA and a tracrRNA.
  • the RNA guide is an engineered construct that includes a tracrRNA and a crRNA (in a single RNA guide).
  • Sequences for RNA guides from multiple CRISPR systems are known in the art and can be searched using public databases (see, e.g., Grissa et al. (2007) Nucleic Acids Res. 35 (web server issue): W52-7; Grissa et al. (2007) BMC Bioinformatics 8: 172; Grissa et al. (2008) Nucleic Acids Res. 36 (web server issue): W145-8; and Moller and Liang (2017) PeerJ 5: e3788; see also the CRISPR database available at:
  • the CRISPR systems described herein include at least one crRNA or a nucleic acid encoding at least one crRNA.
  • the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence, which is typical of precursor crRNA (pre-crRNA) configurations in other CRISPR systems.
  • the crRNA includes a truncated direct repeat sequence and a spacer sequence, which is typical of processed or mature crRNA.
  • the CRISPR-associated protein is capable of cleaving pre-crRNA to form processed or mature crRNA.
  • the CRISPR-associated protein forms a complex with the mature crRNA, and the spacer sequence directs the complex to a sequence-specific binding with the target nucleic acid that is complementary to the spacer sequence.
  • the resulting complex comprises the CRISPR-associated protein and the mature crRNA bound to the target RNA.
  • the CRISPR systems described herein include a mature crRNA. In some embodiments, the CRISPR systems described herein include a pre-crRNA.
  • the CRISPR systems described herein include a plurality of crRNAs (e.g., 2, 3, 4, 5, 10, 15, or more) or a plurality of nucleic acids encoding a plurality of crRNAs.
  • the crRNAs described herein include a direct repeat sequence and a spacer sequence.
  • the crRNA includes, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence.
  • the CRISPR system described herein includes an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide.
  • the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and ⁇ is A or G.
  • the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and ⁇ is A or G .
  • RNA guide direct repeat sequences and effector protein pairs are provided in Table 3.
  • the direct repeat sequence comprises or consists of a nucleic acid sequence listed in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589).
  • the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial three 5' nucleotides.
  • the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial four 5' nucleotides.
  • the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial five 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial six 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial seven 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial eight 5' nucleotides.
  • the direct repeat sequence comprises or consists of the nucleic acid sequence 5'-GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 34) or 5'-CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 72). In some embodiments, the direct repeat sequence comprises or consists of the nucleic acid sequence 5'-CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'-CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the CRISPR-associated protein comprises the amino acid sequence of SEQ ID NO: 1 and the crRNA comprises a direct repeat sequence, wherein the direct repeat sequence comprises or consists of the nucleic acid sequence 5'- GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 34) or 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152).
  • the CRISPR-associated protein comprises the amino acid sequence of SEQ ID NO: 2 and the crRNA comprises a direct repeat sequence, wherein the direct repeat sequence comprises or consists of the nucleic acid sequence 5'- CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 72) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
  • the CRISPR systems described herein include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more) RNA guides.
  • the CRISPR systems described herein include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem.
  • the single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof.
  • Type VI-D CRISPR-Cas effector proteins described herein enables these effectors to be able to target multiple target nucleic acids (e.g., target RNAs) without a loss of activity.
  • the Type VI-D CRISPR-Cas effector proteins may be delivered in complex with multiple RNA guides directed to different target nucleic acids.
  • the Type VI-D CRISPR-Cas effector proteins may be co-delivered with multiple RNA guides, each specific for a different target nucleic acid. Methods of multiplexing using CRISPR-associated proteins are described, for example, in US 9,790, 490 B2, and EP 3009511 Bl, the entire contents of each of which are expressly incorporated herein by reference.
  • the spacer length of crRNAs can range from about 15 to 50 nucleotides.
  • the spacer length of an RNA guide is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides.
  • the spacer length is from 15 to 17 nucleotides (e.g., 15, 16, or 17 nucleotides), from 17 to 20 nucleotides (e.g., 17, 18, 19, or 20 nucleotides), from 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), from 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), from 24 to 27 nucleotides, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides), from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides), or longer.
  • 15 to 17 nucleotides e.g., 15,
  • the direct repeat length of the RNA guide is at least 16 nucleotides, or is from 16 to 20 nucleotides (e.g., 16, 17, 18, 19, or 20 nucleotides). In some embodiments, the spacer length is from about 15 to about 42 nucleotides. In some embodiments, the direct repeat length of the RNA guide is 19 nucleotides.
  • the crRNA sequences can be modified in a manner that allows for formation of a complex between the crRNA and CRISPR-associated protein and successful binding to the target, while at the same time not allowing for successful nuclease activity (i.e., without nuclease activity / without causing indels).
  • These modified guide sequences are referred to as "dead crRNAs," “dead guides,” or “dead guide sequences.”
  • These dead guides or dead guide sequences may be catalytically inactive or conformationally inactive with regard to nuclease activity. Dead guide sequences are typically shorter than respective guide sequences that result in active RNA cleavage.
  • dead guides are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective RNA guides that have nuclease activity.
  • Dead guide sequences of RNA guides can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length), from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length).
  • the disclosure provides non-naturally occurring or engineered CRISPR systems including a functional CRISPR-associated protein as described herein, and a crRNA, wherein the crRNA comprises a dead crRNA sequence whereby the crRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a genomic locus of interest in a cell without detectable nuclease activity (e.g., RNAse activity).
  • a functional CRISPR-associated protein as described herein and a crRNA
  • the crRNA comprises a dead crRNA sequence whereby the crRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a genomic locus of interest in a cell without detectable nuclease activity (e.g., RNAse activity).
  • dead guides A detailed description of dead guides is described, e.g., in International Publication No. WO 2016/094872, which is incorporated herein by reference in its entirety.
  • RNA guides can be generated as components of inducible systems.
  • the inducible nature of the systems allows for spatio-temporal control of gene editing or gene expression.
  • the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.
  • RNA guides e.g., crRNA
  • inducible promoters e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems), and arabinose-inducible gene expression systems.
  • inducible systems include, e.g., small molecule two- hybrid transcription activations systems (FKBP, ABA, etc.), light inducible systems
  • Chemically modified bases such as 2- thiouridine or N6-methyladenosine, among others, can allow for either stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering," Front. Genet., 2012 Aug 20; 3: 154). Additionally, RNA is amenable to both 5' and 3' end conjugations with a variety of functional moieties including fluorescent dyes, polyethylene glycol, or proteins.
  • modifying an oligonucleotide with a 2'-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing.
  • a 2'- OMe modification can affect how the oligonucleotide interacts with transfection reagents, proteins or any other molecules in the cell. The effects of these modifications can be determined by empirical testing.
  • the crRNA includes one or more phosphorothioate modifications. In some embodiments, the crRNA includes one or more locked nucleic acids for the purpose of enhancing base pairing and/or increasing nuclease resistance.
  • RNA guides e.g., crRNAs
  • the optimized length of an RNA guide can be determined by identifying the processed form of crRNA (i.e., a mature crRNA), or by empirical length studies for crRNA tetraloops.
  • the crRNAs can also include one or more aptamer sequences.
  • Aptamers are oligonucleotide or peptide molecules have a specific three-dimensional structure and can bind to a specific target molecule.
  • the aptamers can be specific to gene effectors, gene activators, or gene repressors.
  • the aptamers can be specific to a protein, which in turn is specific to and recruits and/or binds to specific gene effectors, gene activators, or gene repressors.
  • the effectors, activators, or repressors can be present in the form of fusion proteins.
  • the RNA guide has two or more aptamer sequences that are specific to the same adaptor proteins.
  • the two or more aptamer sequences are specific to different adaptor proteins.
  • the adaptor proteins can include, e.g., MS2, PP7, QP, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇
  • the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein.
  • the aptamer sequence is a MS2 binding loop (5'- ggcccAACAUGAGGAUCACCCAUGUCUGCAGgggcc-3' (SEQ ID NO: 169)).
  • the apatamer sequence is a QBeta binding loop (5'- ggcccAUGCUGUCUAAGACAGCAUgggcc-3' (SEQ ID NO: 170)).
  • the aptamer sequence is a PP7 binding loop (5'- ggcccUAAGGGUUUAUAUGGAAACCCUUAgggcc-3' (SEQ ID NO: 173)).
  • the target nucleic acids can be a DNA molecule or a RNA molecule.
  • the CRISPR-associated proteins described herein have RNAse activity.
  • the target nucleic acids can be any RNA molecule of interest, including naturally-occurring and engineered RNA molecules.
  • the target RNA can be an mRNA, a tRNA, a ribosomal RNA (rRNA), a microRNA (miRNA), an interfering RNA (siRNA), a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA.
  • the target nucleic acid is associated with a condition or disease (e.g., an infectious disease or a cancer).
  • a condition or disease e.g., an infectious disease or a cancer
  • the systems described herein can be used to treat a condition or disease by targeting these nucleic acids.
  • the target nucleic acid associated with a condition or disease may be an RNA molecule that is overexpressed in a diseased cell (e.g., a cancer or tumor cell).
  • the target nucleic acid may also be a toxic RNA and/or a mutated RNA (e.g., an mRNA molecule having a splicing defect or a mutation).
  • the target nucleic acid may also be an RNA that is specific for a particular microorganism (e.g., a pathogenic bacteria).
  • the degree of complementarity between a guide sequence e.g., a crRNA
  • its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of
  • RNA guides can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • mutations can be introduced to the CRISPR systems so that the CRISPR systems can distinguish between target and off-target sequences that have greater than 80%, 85%, 90%, or 95% complementarity.
  • the degree of complementarity is from 80% to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches).
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%.
  • the degree of complementarity is 100%. It is known in the field that complete complementarity is not required, provided there is sufficient complementarity to be functional. Modulations of cleavage efficiency can be exploited by introduction of mismatches, e.g., one or more mismatches, such as 1 or 2 mismatches between spacer sequence and target sequence, including the position of the mismatch along the spacer/target.
  • a mismatch e.g., a double mismatch
  • cleavage efficiency can be modulated. For example, if less than 100% cleavage of targets is desired (e.g., in a cell population), 1 or 2 mismatches between spacer and target sequence can be introduced in the spacer sequences.
  • the CRISPR systems described herein further comprise a target nucleic acid (e.g., a linear or circular nucleic acid) which may advantageously be used to activate the collateral RNAse activity of a Type VI-D CRISPR-Cas effector protein in a controlled manner.
  • a target nucleic acid e.g., a linear or circular nucleic acid
  • the activation of the collateral RNAse activity of the effector protein may be controlled.
  • exogenous target nucleic acid may be included in the system to increase the activation rate of the collateral RNAse activity of a Type VI-D CRISPR-Cas effector protein.
  • the target nucleic acid is a DNA molecule.
  • the target nucleic acid is an RNA molecule (e.g., a mRNA molecule).
  • the system includes a DNA molecule (e.g., a plasmid DNA) that codes for the target nucleic acid that is specifically targeted by the Type VI-D CRISPR-Cas effector protein and crRNA complex, operably linked to a promoter.
  • the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter.
  • the CRISPR systems described herein includes at least one accessory protein.
  • the accessory proteins described herein enhance the nuclease activity of CRISPR-associated proteins (e.g., Type VI-D CRISPR-Cas effector proteins) as compared to the nuclease activity of the CRISPR associated protein in the absence of the accessory protein.
  • the ability of the accessory proteins described herein to enhance the nuclease activity of CRISPR-associated proteins is particularly desireable in clinical and therapeutic applications. Therefore, CRISPR systems including at least one accessory protein are provided herein.
  • an accessory protein described herein may be used in combination with CRISPR-associated proteins known in the art in order to enhance their nuclease activity.
  • an accessory protein may be used in combination with a Type VI-D CRISPR-Cas effector protein described herein to enhance its nuclease activity (e.g., collateral RNAse activity or targeted RNAse activity).
  • the accessory protein includes a WYL domain (PFAM:
  • WYL domains are SH3 beta-barrel fold containing domains named for three conserved amino acids found in some domains belonging to the WYL-like superfamily.
  • WYL domain protein, sll7009 has been found to be a negative regulator of the Synechocystis sp. I-D CRISPR-Cas system (see, e.g., Hein et al. (2013) RNA Biol. 10: 852-64).
  • the accessory protein includes at least one WYL domain. In some embodiments, the accessory protein includes two WYL domains. In some
  • the accessory protein includes a helix-turn-helix (HTH) fold. In some embodiments, the accessory protein includes a ribbon-helix-helix (RHH) fold. In some embodiments, the accessory protein includes at least one WYL domain, wherein the WYL domain comprises the amino acid sequence PXXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid.
  • the accessory protein includes at least one WYL domain, wherein the WYL domain comprises the amino acid sequence PXXXiXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid; and at least one ribbon-ribbon-helix (RHH) fold or at least one helix-turn-helix (HTH) domain.
  • the amino acid sequence of the WYL domain is separate from (i.e., does not overlap with) an RHH fold or an HTH fold.
  • the accessory proteins describe herein modulate the RNAse activity of a CRISPR-associated protein.
  • the accessory protein modulates (e.g., increases or decreases) the collateral RNAse activity of a CRISPR-associated protein.
  • the accessory protein modulates (e.g., increases or decreases) the RNA-binding activity of a CRISPR-associated protein.
  • the accessory protein modulates (e.g., increases or decreases) the crRNA processing activity of a CRISPR-associated protein.
  • the accessory protein modulates (e.g., increases or decreases) the targeted RNAse activity of a CRISPR-associated protein.
  • the accessory proteins described herein enhances the RNAse activity of a CRISPR-associated protein (e.g., a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, a Cas9 protein).
  • a CRISPR-associated protein e.g., a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, a Cas9 protein.
  • the accessory protein enhances the collateral RNAse activity of a CRISPR-associated protein.
  • the accessory protein enhances the crRNA processing activity of a CRISPR- associated protein.
  • the accessory protein enhances the RNA-binding activity of a CRISPR-associated protein.
  • the accessory protein enhances the targeted RNAse activity of a CRISPR-associated protein.
  • CRISPR systems comprising an accessory protein described herein are particularly useful in applications where increased sequence-specific or collateral RNA degradation is desireable. For example, in diagnostic applications, enhanced RNAse activity provides a greater degree of sensitivity, allowing the detection of lower concentrations of a target RNA.
  • an accessory protein described herein enhances the RNAse activity of the ternary complex of multiple CRISPR Type VI effectors. The ability of the accessory protein to enhance the RNAse of multiple effectors is particularly useful in applications where combinations of Type VI effectors of different sub-types are used together, for example in multi-channel diagnostic applications.
  • the accessory protein can enhance the RNAse activity of Type VI effectors outside the Casl3d family thereby providing a valuable tool for screening the activity of uncharacterized Type VI effectors.
  • accessory proteins are provided below in Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671).
  • the accessory proteins include an amino acid sequence having at least about 80% identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of any one of Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671).
  • the accessory protein includes the amino acid sequence of any one of the proteins in Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671).
  • the accessory protein is RspWYLl (SEQ ID NO: 81).
  • the CRISPR systems described herein have a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, or activating) a target polynucleotide or nucleic acid in a multiplicity of cell types.
  • the CRISPR systems have a broad spectrum of applications in, e.g., DNA/RNA detection (e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK)), tracking and labeling of nucleic acids, enrichment assays (extracting desired sequence from background), controlling interfering RNA or miRNA, detecting circulating tumor DNA, preparing next generation library, drug screening, disease diagnosis and prognosis, and treating various genetic disorders.
  • DNA/RNA detection e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK)
  • SHERLOCK specific high sensitivity enzymatic reporter unlocking
  • enrichment assays extract desired sequence from background
  • controlling interfering RNA or miRNA detecting circulating tumor DNA
  • the CRISPR systems described herein can be used in DNA or RNA detection.
  • CRISPR-associated proteins can be reprogrammed with CRISPR RNAs (crRNAs) to provide a platform for specific RNA sensing.
  • crRNAs CRISPR RNAs
  • activated CRISPR-associated proteins engage in "collateral" cleavage of nearby non-targeted RNAs. This crRNA-programmed collateral cleavage activity allows the CRISPR systems to detect the presence of a specific RNA by triggering programmed cell death or by nonspecific degradation of labeled RNA.
  • UnLOCKing provides an in vitro nucleic acid detection platform with attomolar sensitivity based on nucleic acid amplification and collateral cleavage of a reporter RNA, allowing for real-time detection of the target.
  • the detection can be combined with different isothermal amplification steps.
  • recombinase polymerase amplification RPA
  • T7 transcription to convert amplified DNA to RNA for subsequent detection.
  • SHERLOCK amplification by RPA, T7 RNA polymerase transcription of amplified DNA to RNA, and detection of target RNA by collateral RNA cleavage-mediated release of reporter signal.
  • the CRISPR-associated proteins can further be used in Northern blot assays, which use electrophoresis to separate RNA samples by size.
  • the CRISPR-associated proteins can be used to specifically bind and detect the target RNA sequence.
  • the CRISPR-associated proteins can also be fused to a fluorescent protein (e.g., GFP) and used to track RNA localization in living cells. More particularly, the CRISPR-associated proteins can be inactivated in that they no longer cleave RNAs as described above.
  • CRISPR-associated proteins can be used to determine the localization of the RNA or specific splice variants, the level of mRNA transcripts, up- or down- regulation of transcripts and disease-specific diagnosis.
  • the CRISPR-associated proteins can be used for visualization of RNA in (living) cells using, for example, fluorescent microscopy or flow cytometry, such as fluorescence- activated cell sorting (FACS), which allows for high-throughput screening of cells and recovery of living cells following cell sorting.
  • fluorescent microscopy or flow cytometry such as fluorescence- activated cell sorting (FACS)
  • FACS fluorescence- activated cell sorting
  • the CRISPR systems described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH). These methods are described in, e.g., Chen et al, "Spatially resolved, highly multiplexed RNA profiling in single cells," Science, 2015 Apr 24; 348(6233):aaa6090, which is incorporated herein by reference herein in its entirety.
  • MEFISH multiplexed error-robust fluorescence in situ hybridization
  • the CRISPR systems described herein can be used to detect a target RNA in a sample (e.g., a clinical sample, a cell, or a cell lysate).
  • a sample e.g., a clinical sample, a cell, or a cell lysate.
  • the collateral RNAse activity of the Type VI-D CRISPR-Cas effector proteins described herein is activated when the effector proteins bind to a target nucleic acid.
  • the effector protein cleaves a labeled detector RNA to generate a signal (e.g., an increased signal or a decreased signal) thereby allowing for the qualitative and quantitative detection of the target RNA in the sample.
  • the specific detection and quantification of RNA in the sample allows for a multitude of applications including diagnostics.
  • the methods include contacting a sample with: i) an RNA guide (e.g., crRNA) and/or a nucleic acid encoding the RNA guide, wherein the RNA guide consists of a direct repeat sequence and a spacer sequence capable of hybridizing to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein and/or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single- stranded target RNA in the sample.
  • an RNA guide e.g
  • the methods further comprise comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
  • the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
  • the labeled detector RNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair.
  • FRET fluorescence resonance energy transfer
  • an amount of detectable signal produced by the labeled detector RNA is decreased or increased.
  • the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein. In some embodiments, a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein. In some embodiments, the labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof.
  • the methods include the multi-channel detection of multiple independent target RNAs in a sample (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more target RNAs) by using multiple Type VI-D CRISPR-Cas systems, each including a distinct orthologous effector protein and corresponding RNA guides, allowing for the differentiation of multiple target RNAs in the sample.
  • the methods include the multi-channel detection of multiple independent target RNAs in a sample, with the use of multiple instances of Type VI-D CRISPR-Cas systems, each containing an orthologous effector protein with differentiable collateral RNAse substrates. Methods of detecting an RNA in a sample using CRISPR-associated proteins are described, for example, in U.S. Patent Publication No. 2017/0362644, the entire contents of which are incorporated herein by reference.
  • In vitro proximity labeling techniques employ an affinity tag combined with, a reporter group, e.g., a photoactivatable group, to label polypeptides and RNAs in the vicinity of a protein or RNA of interest in vitro. After UV irradiation, the photoactivatable groups react with proteins and other molecules that are in close proximity to the tagged molecules, thereby labelling them. Labelled interacting molecules can
  • the CRISPR-associated proteins can for instance be used to target probes to selected RNA sequences. These applications can also be applied in animal models for in vivo imaging of diseases or difficult-to culture cell types. The methods of tracking and labeling of nucleic acids are described, e.g., in US 8795965, WO
  • the CRISPR systems e.g., CRISPR-associated proteins
  • CRISPR-associated proteins can be used to isolate and/or purify the RNA.
  • the CRISPR-associated proteins can be fused to an affinity tag that can be used to isolate and/or purify the RNA-CRISPR-associated protein complex. These applications are useful, e.g., for the analysis of gene expression profiles in cells.
  • the CRISPR-associated proteins can be used to target a specific noncoding RNA (ncRNA) thereby blocking its activity.
  • ncRNA noncoding RNA
  • the CRISPR-associated proteins can be used to specifically enrich a particular RNA (including but not limited to increasing stability, etc.), or alternatively, to specifically deplete a particular RNA (e.g., particular splice variants, isoforms, etc.).
  • the CRISPR systems described herein can be used for preparing next generation sequencing (NGS) libraries.
  • NGS next generation sequencing
  • the CRISPR systems can be used to disrupt the coding sequence of a target gene, and the CRISPR- associated protein transfected clones can be screened simultaneously by next-generation sequencing (e.g., on the Ion Torrent PGM system).
  • next-generation sequencing e.g., on the Ion Torrent PGM system.
  • Microorganisms e.g., E. coli, yeast, and microalgae
  • E. coli, yeast, and microalgae are widely used for synthetic biology.
  • the development of synthetic biology has a wide utility, including various clinical applications.
  • the programmable CRISPR systems can be used to split proteins of toxic domains for targeted cell death, e.g., using cancer-linked RNA as target transcript.
  • pathways involving protein-protein interactions can be influenced in synthetic biological systems with e.g. fusion complexes with the appropriate effectors such as kinases or enzymes.
  • crRNAs that target phage sequences can be introduced into the microorganism.
  • the disclosure also provides methods of vaccinating a microorganism (e.g., a production strain) against phage infection.
  • the CRISPR systems provided herein can be used to engineer microorganisms, e.g., to improve yield or improve fermentation efficiency.
  • the CRISPR systems described herein can be used to engineer microorganisms, such as yeast, to generate biofuel or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars.
  • the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and/or to modify endogenous genes, which may interfere with the biofuel synthesis.
  • the CRISPR systems provided herein can be used to induce death or dormancy of a cell (e.g., a microorganism such as an engineered microorganism). These methods can be used to induce dormancy or death of a multitude of cell types including prokaryotic and eukaryotic cells, including, but not limited to mammalian cells (e.g., cancer cells, or tissue culture cells), protozoans, fungal cells, cells infected with a virus, cells infected with an intracellular bacteria, cells infected with an intracellular protozoan, cells infected with a prion, bacteria (e.g., pathogenic and non-pathogenic bacteria), protozoans, and unicellular and multicellular parasites.
  • mammalian cells e.g., cancer cells, or tissue culture cells
  • protozoans fungal cells
  • cells infected with a virus e.g., cells infected with an intracellular bacteria
  • engineered microorganisms e.g., bacteria
  • the systems described herein can be used as "kill-switches” to regulate and/or prevent the propagation or dissemination of an engineered microorganism.
  • the systems described herein can also be used in applications where it is desirable to kill or control a specific microbial population (e.g., a bacterial population).
  • the systems described herein may include an RNA guide (e.g., a crRNA) that targets a nucleic acid (e.g., an RNA) that is genus-, species-, or strain- specific, and can be delivered to the cell.
  • a nucleic acid e.g., an RNA
  • the collateral RNAse activity of the Type VI-D CRISPR-Cas effector proteins is activated leading to the cleavage of non-target RNA within the microorganisms, ultimately resulting in dormancy or death.
  • the methods comprise contacting the cell with a system described herein including a Type VI-D CRISPR-Cas effector proteins or a nucleic acid encoding the effector protein, and a RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the spacer sequence is complementary to at least 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides ) of a target nucleic acid (e.g., a genus-, strain-, or species-specific RNA guide).
  • a target nucleic acid e.g., a genus-, strain-, or species-specific RNA guide.
  • the cleavage of non-target RNA by the Type VI-D CRISPR-Cas effector proteins may induce programmed cell death, cell toxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, a reduction of cell growth, or a reduction in cell proliferation.
  • the cleavage of non- target RNA by the Type VI-D CRISPR-Cas effector proteins may be bacteriostatic or bacteriocidal.
  • the CRISPR systems described herein have a wide variety of utility in plants.
  • the CRISPR systems can be used to engineer genomes of plants (e.g., improving production, making products with desired post-translational modifications, or introducing genes for producing industrial products).
  • the CRISPR systems can be used to introduce a desired trait to a plant (e.g., with or without heritable modifications to the genome), or regulate expression of endogenous genes in plant cells or whole plants.
  • the CRISPR systems can be used to identify, edit, and/or silence genes encoding specific proteins, e.g., allergenic proteins (e.g., allergenic proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans).
  • Gene drive is the phenomenon in which the inheritance of a particular gene or set of genes is favorably biased.
  • the CRISPR systems described herein can be used to build gene drives.
  • the CRISPR systems can be designed to target and disrupt a particular allele of a gene, causing the cell to copy the second allele to fix the sequence. Because of the copying, the first allele will be converted to the second allele, increasing the chance of the second allele being transmitted to the offspring.
  • a detailed method regarding how to use the CRISPR systems described herein to build gene drives is described, e.g., in Hammond et al, "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae " Nat. Biotechnol., 2016 Jan; 34(l):78-83, which is incorporated herein by reference in its entirety.
  • pooled CRISPR screening is a powerful tool for identifying genes involved in biological mechanisms such as cell proliferation, drug resistance, and viral infection.
  • Cells are transduced in bulk with a library of RNA guide-encoding vectors described herein, and the distribution of RNA guides is measured before and after applying a selective challenge.
  • Pooled CRISPR screens work well for mechanisms that affect cell survival and proliferation, and they can be extended to measure the activity of individual genes (e.g., by using engineered reporter cell lines).
  • Arrayed CRISPR screens in which only one gene is targeted at a time, make it possible to use RNA-seq as the readout.
  • the CRISPR systems as described herein can be used in single-cell CRISPR screens.
  • RNA guide library can be used to perform in situ saturating mutagenesis for particular genes or regulatory elements.
  • Such methods can reveal critical minimal features and discrete vulnerabilities of these genes or regulatory elements (e.g., enhancers). These methods are described, e.g., in Canver et al, "BCL11 A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, 2015 Nov 12;
  • the CRISPR systems described herein can have various RNA-related applications, e.g., modulating gene expression, degrading a RNA molecule, inhibiting RNA expression, screening RNA or RNA products, determining functions of lincRNA or non-coding RNA, inducing cell dormancy, inducing cell cycle arrest, reducing cell growth and/or cell proliferation, inducing cell anergy, inducing cell apoptosis, inducing cell necrosis, inducing cell death, and/or inducing programmed cell death.
  • WO 2016/205764 Al which is incorporated herein by reference in its entirety.
  • the methods described herein can be performed in vitro, in vivo, or ex vivo.
  • the CRISPR systems described herein can be administered to a subject having a disease or disorder to target and induce cell death in a cell in a diseased state (e.g., cancer cells or cells infected with an infectious agent).
  • a diseased state e.g., cancer cells or cells infected with an infectious agent.
  • the CRISPR systems described herein can be used to target and induce cell death in a cancer cell, wherein the cancer cell is from a subject having a Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia
  • the CRISPR systems described herein can be used to modulate gene expression.
  • the CRISPR systems can be used, together with suitable RNA guides, to target gene expression, via control of RNA processing.
  • the control of RNA processing can include, e.g., RNA processing reactions such as RNA splicing (e.g., alternative splicing), viral replication, and tRNA biosynthesis.
  • the RNA targeting proteins in combination with suitable RNA guides can also be used to control RNA activation (RNAa).
  • RNA activation is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the
  • RNAa leads to the promotion of gene expression, so control of gene expression may be achieved that way through disruption or reduction of RNAa.
  • the methods include the use of the RNA targeting CRISPR as substitutes for e.g., interfering ribonucleic acids (such as siRNAs, shRNAs, or dsRNAs).
  • interfering ribonucleic acids such as siRNAs, shRNAs, or dsRNAs.
  • the target RNAs can include interfering RNAs, i.e., RNAs involved in the RNA interference pathway, such as small hairpin RNAs (shRNAs), small interfering (siRNAs), etc.
  • the target RNAs include, e.g., miRNAs or double stranded RNAs (dsRNA).
  • RNA targeting protein and suitable RNA guides are selectively expressed (for example spatially or temporally under the control of a regulated promoter, for example a tissue- or cell cycle-specific promoter and/or enhancer), this can be used to protect the cells or systems (in vivo or in vitro) from RNA interference (RNAi) in those cells.
  • a regulated promoter for example a tissue- or cell cycle-specific promoter and/or enhancer
  • RNAi RNA interference
  • This may be useful in neighboring tissues or cells where RNAi is not required or for the purposes of comparison of the cells or tissues where the CRISPR-associated proteins and suitable crRNAs are and are not expressed (i.e., where the RNAi is not controlled and where it is, respectively).
  • RNA targeting proteins can be used to control or bind to molecules comprising or consisting of RNAs, such as ribozymes, ribosomes, or riboswitches.
  • the RNA guides can recruit the RNA targeting proteins to these molecules so that the RNA targeting proteins are able to bind to them.
  • Riboswitches are regulatory segments of messenger RNAs that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules.
  • a specific riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene.
  • the riboswitch activity can be controlled by the use of the RNA targeting proteins in combination with suitable RNA guides to target the riboswitches. This may be achieved through cleavage of, or binding to, the riboswitch.
  • the CRISPR-associated proteins described herein can be fused to a base-editing domain, such as ADAR1, ADAR2, APOBEC, or activation-induced cytidine deaminase (AID), and can be used to modify an RNA sequence (e.g., an mRNA).
  • a base-editing domain such as ADAR1, ADAR2, APOBEC, or activation-induced cytidine deaminase (AID)
  • AID activation-induced cytidine deaminase
  • the CRISPR-associated protein includes one or more mutations (e.g., in a catalytic domain), which renders the CRISPR-associated protein incapable of cleaving RNA.
  • the CRISPR-associated proteins can be used with an RNA- binding fusion polypeptide comprising a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID) fused to an RNA-binding domain, such as MS2 (also known as MS2 coat protein), Qbeta (also known as Qbeta coat protein), or PP7 (also known as PP7 coat protein).
  • a base-editing domain e.g., ADAR1, ADAR2, APOBEC, or AID
  • RNA-binding domain such as MS2 (also known as MS2 coat protein), Qbeta (also known as Qbeta coat protein), or PP7 (also known as PP7 coat protein).
  • MS2 also known as MS2 coat protein
  • Qbeta also known as Qbeta coat protein
  • PP7 also known as PP7 coat protein
  • MS2 (MS2 coat protein)
  • the RNA binding domain can bind to a specific sequence (e.g., an aptamer sequence) or secondary structure motifs on a crRNA of the system described herein (e.g., when the crRNA is in an effector-crRNA complex), thereby recruiting the RNA binding fusion polypeptide (which has a base-editing domain) to the effector complex.
  • a specific sequence e.g., an aptamer sequence
  • secondary structure motifs on a crRNA of the system described herein (e.g., when the crRNA is in an effector-crRNA complex)
  • the CRISPR system includes a CRISPR associated protein, a crRNA having an aptamer sequence (e.g., an MS2 binding loop, a QBeta binding loop, or a PP7 binding loop), and a RNA-binding fusion polypeptide having a base-editing domain fused to an RNA-binding domain that specifically binds to the aptamer sequence.
  • the CRISPR-associated protein forms a complex with the crRNA having the aptamer sequence.
  • the RNA-binding fusion polypeptide binds to the crRNA (via the aptamer sequence) thereby forming a tripartite complex that can modify a target RNA.
  • an inactivated CRISPR-associated protein described herein e.g., a CRISPR associated protein having one or more mutations in a catalytic domain
  • a CRISPR associated protein having one or more mutations in a catalytic domain can be used to target and bind to specific splicing sites on RNA transcripts. Binding of the inactivated CRISPR-associated protein to the RNA may sterically inhibit interaction of the spliceosome with the transcript, enabling alteration in the frequency of generation of specific transcript isoforms.
  • Methods of using CRISPR systems to alter splicing are described, e.g., in International Publication No. WO 2017/219027, which is incorporated herein by reference in its entirety, and in particular with respect to its discussion of RNA splicing.
  • the CRISPR systems described herein can have various therapeutic applications.
  • the new CRISPR systems can be used to treat various diseases and disorders, e.g., genetic disorders (e.g., monogenetic diseases), diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne Muscular Dystrophy (DMD), BCL1 la targeting), and various cancers, etc.
  • diseases and disorders e.g., genetic disorders (e.g., monogenetic diseases), diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne Muscular Dystrophy (DMD), BCL1 la targeting), and various cancers, etc.
  • the CRISPR systems described herein can be used to edit a target nucleic acid to modify the target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleic acid residues).
  • the CRISPR systems described herein comprise an exogenous donor template nucleic acid (e.g., a DNA molecule or a RNA molecule), which comprises a desirable nucleic acid sequence.
  • an exogenous donor template nucleic acid e.g., a DNA molecule or a RNA molecule
  • the molecular machinery of the cell will utilize the exogenous donor template nucleic acid in repairing and/or resolving the cleavage event.
  • the molecular machinery of the cell can utilize an endogenous template in repairing and/or resolving the cleavage event.
  • the CRISPR systems described herein may be used to alter a target nucleic acid resulting in an insertion, a deletion, and/or a point mutation).
  • the insertion is a scarless insertion (i.e., the insertion of an intended nucleic acid sequence into a target nucleic acid resulting in no additional unintended nucleic acid sequence upon resolution of the cleavage event).
  • Donor template nucleic acids may be double stranded or single stranded nucleic acid molecules (e.g., DNA or RNA). Methods of designing exogenous donor template nucleic acids are described, for example, in International Publication No. WO 2016/094874 Al, the entire contents of which are expressly incorporated herein by reference.
  • the CRISPR systems described herein can be used for treating a disease caused by overexpression of RNAs, toxic RNAs, and/or mutated RNAs (e.g., splicing defects or truncations).
  • expression of toxic RNAs may be associated with the formation of nuclear inclusions and late-onset degenerative changes in brain, heart, or skeletal muscle.
  • the disorder is myotonic dystrophy. In myotonic dystrophy, the main pathogenic effect of the toxic RNAs is to sequester binding proteins and compromise the regulation of alternative splicing (see, e.g., Osborne et al, "RNA-dominant diseases," Hum. Mol.
  • DM dystrophia my otonica
  • UTR 3 '-untranslated region
  • DMPK a gene encoding a cytosolic protein kinase.
  • the CRISPR systems as described herein can target overexpressed RNA or toxic RNA, e.g., the DMPK gene or any of the mis-regulated alternative splicing in DM1 skeletal muscle, heart, or brain.
  • the CRISPR systems described herein can also target trans-acting mutations affecting RNA- dependent functions that cause various diseases such as, e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA), and Dyskeratosis congenita.
  • various diseases such as, e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA), and Dyskeratosis congenita.
  • SMA Spinal muscular atrophy
  • Dyskeratosis congenita e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA), and Dyskeratosis congenita.
  • SMA Spinal muscular atrophy
  • Dyskeratosis congenita e.g., Dyskeratosis congenita.
  • a list of diseases that can be treated using the CRISPR systems described herein is summarized in Cooper et al, "RNA and disease," Cell, 136.4 (2009): 777-793, and WO 2016/2057
  • the CRISPR systems described herein can also be used in the treatment of various tauopathies, including, e.g., primary and secondary tauopathies, such as primary age-related tauopathy (PART)/Neurofibrillary tangle (NFT)-predominant senile dementia (with NFTs similar to those seen in Alzheimer Disease (AD), but without plaques), dementia pugilistica (chronic traumatic encephalopathy), and progressive supranuclear palsy.
  • PART primary age-related tauopathy
  • NFT Neurofibrillary tangle
  • a useful list of tauopathies and methods of treating these diseases are described, e.g., in WO 2016205764, which is incorporated herein by reference in its entirety.
  • the CRISPR systems described herein can also be used to target mutations disrupting the cis-acting splicing codes that can cause splicing defects and diseases.
  • diseases include, e.g., motor neuron degenerative disease that results from deletion of the SMNl gene (e.g., spinal muscular atrophy), Duchenne Muscular Dystrophy (DMD), frontotemporal dementia, and Parkinsonism linked to chromosome 17 (FTDP-17), and cystic fibrosis.
  • the CRISPR systems described herein can further be used for antiviral activity, in particular against RNA viruses.
  • the CRISPR-associated proteins can target the viral RNAs using suitable RNA guides selected to target viral RNA sequences.
  • the CRISPR systems described herein can also be used to treat a cancer in a subject (e.g., a human subject).
  • a subject e.g., a human subject
  • the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule that is aberrant (e.g., comprises a point mutation or are alternatively-spliced) and found in cancer cells to induce cell death in the cancer cells (e.g., via apoptosis).
  • the CRISPR systems described herein can also be used to treat an infectious disease in a subject.
  • the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule expressed by an infectious agent (e.g., a bacteria, a virus, a parasite or a protozoan) in order to target and induce cell death in the infectious agent cell.
  • an infectious agent e.g., a bacteria, a virus, a parasite or a protozoan
  • the CRISPR systems may also be used to treat diseases where an intracellular infectious agent infects the cells of a host subject.
  • By programming the CRISPR-associated protein to target a RNA molecule encoded by an infectious agent gene cells infected with the infectious agent can be targeted and cell death induced.
  • RNA sensing assays can be used to detect specific RNA substrates.
  • the CRISPR-associated proteins can be used for RNA-based sensing in living cells. Examples of applications are diagnostics by sensing of, for examples, disease-specific RNAs.
  • the CRISPR systems described herein, or components thereof, nucleic acid molecules thereof, and/or nucleic acid molecules encoding or providing components thereof can be delivered by various delivery systems such as vectors, e.g., plasmids and viral delivery vectors (e.g., adeno-associated virus AAV vectors).
  • vectors e.g., plasmids and viral delivery vectors (e.g., adeno-associated virus AAV vectors).
  • the CRISPR-associated proteins and/or any of the RNAs (e.g., RNA guides) and/or accessory proteins can be delivered using suitable vectors, e.g., plasmids or viral vectors, such as adeno-associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or combinations thereof.
  • AAV adeno-associated viruses
  • the proteins and one or more crRNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors.
  • the nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to the bacteria using a phage.
  • Exemplary phages include, but are not limited to, T4 phage, Mu, ⁇ phage, T5 phage, T7 phage, T3 phage, ⁇ 29, M13, MS2, Q , and ⁇ 174.
  • the vectors e.g., plasmids or viral vectors
  • the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration.
  • Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
  • the delivery is via adenoviruses, which can be at a single dose containing at least 1 x 10 5 particles (also referred to as particle units, pu) of
  • the dose preferably is at least about 1 x 10 6 particles, at least about 1 x 10 7 particles, at least about 1 x 10 8 particles, and at least about 1 x 10 9 particles of the adenoviruses.
  • Exemplary delivery methods and the doses are described, e.g., in WO 2016205764 Al and U.S. Patent No. 8,454,972 B2, both of which are incorporated herein by reference in the entirety.
  • the delivery is via a recombinant adeno-associated virus (rAAV) vector.
  • a modified AAV vector may be used for delivery.
  • Modified AAV vectors can be based on one or more of several capside types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV 8.2. AAV9, AAV rhlO, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV 6) and pseudotyped AAV (e.g., AAV2/8, AAV2/5 and AAV2/6).
  • modified AAV vectors and techniques that may be used to produce rAAV particles are known in the art (see, e.g.
  • the delivery is via plasmids.
  • the dosage can be a sufficient number of plasmids to elicit a response.
  • suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg.
  • Plasmids will generally include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR-associated proteins and/or an accessory protein, each operably linked to a promoter (e.g., the same promoter or a different promoter); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii).
  • the plasmids can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on different vectors.
  • the frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or a person skilled in the art.
  • the delivery is via liposomes or lipofection formulations and the like, and can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Pat. Nos. 5,593,972; 5,589,466; and 5,580,859; each of which is incorporated herein by reference in its entirety.
  • the delivery is via nanoparticles or exosomes.
  • exosomes have been shown to be particularly useful in delivery RNA.
  • CRISPR-associated proteins are linked to the CRISPR-associated proteins.
  • the CRISPR-associated proteins and/or RNA guides are coupled to one or more CPPs to effectively transport them inside cells (e.g., plant protoplasts).
  • the CRISPR- associated proteins and/or RNA guides are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cell delivery.
  • CPPs are short peptides of fewer than 35 amino acids derived either from proteins or from chimeric sequences capable of transporting biomolecules across cell membrane in a receptor independent manner.
  • CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having proline- rich and anti-microbial sequences, and chimeric or bipartite peptides.
  • CPPs include, e.g., Tat (which is a nuclear transcriptional activator protein required for viral replication by HIV type 1), penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin ⁇ 3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide.
  • Tat which is a nuclear transcriptional activator protein required for viral replication by HIV type 1
  • FGF Kaposi fibroblast growth factor
  • FGF Kaposi fibroblast growth factor
  • integrin ⁇ 3 signal peptide sequence integrin ⁇ 3 signal peptide sequence
  • polyarginine peptide Args sequence e.g., in Hallbrink et al, "Prediction of cell-penetrating peptides," Methods Mol.
  • the disclosure relates to the use of computational methods and algorithms to search for and identify novel protein families that exhibit a strong cooccurrence pattern with certain other features within naturally occurring genome sequences.
  • these computational methods are directed to identifying protein families that co-occur in close proximity to CRISPR arrays.
  • the methods disclosed herein are useful in identifying proteins that naturally occur within close proximity to other features, both non-coding and protein-coding (for example, CRISPR Casl proteins). It should be understood that the methods and calculations described herein may be performed on one or more computing devices.
  • a set of genomic sequences are obtained from genomic or metagenomie databases.
  • the databases comprise short reads, contig level data, assembled scaffolds, or complete organisms.
  • the database may comprise genomic sequence data from prokaryotic organisms, or eukaryotic organisms, or may include data from metagenomie environmental samples.
  • Exemplary database repositories include NCBI RefSeq, NCBI GenBank, NCBI Whole Genome Shotgun (WGS), and JGI integrated Microbial Genomes (IMG).
  • a minimum size requirement is imposed to select genome sequence data of a specified minimum length.
  • the minimum contig length may be 100 nucleotides, 500 nt, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 40 kb, or 50 kb.
  • the GeneMark Suite is used to predict proteins from genome sequences.
  • Prodigal is used to predict proteins from genome sequences.
  • multiple protein prediction algorithms may be used over the same set of sequence data with the resulting set of proteins de-duplicated.
  • CRISPR arrays are identified from the genome sequence data.
  • PILER-CR is used to identity CRISPR arrays.
  • CRISPR Recognition Tool is used to identify CR1SPR arrays.
  • multiple CRISPR array identification tools may be used over the same set of sequence data with the resulting set of CRISPR arrays de-duplicated.
  • proteins in close proximity to CRISPR. arrays are identified.
  • proximity is defined as a nucleotide distance, and may be within 20 kb, 15 kb, or 5 kb.
  • proximity is defined as the number of open reading frames (ORFs) between a protein and a CRISPR array, and certain exemplar ⁇ ' distances may be 10, 5, 4, 3, 2, 1, or 0 ORFs.
  • ORFs open reading frames
  • the proteins identified as being within close proximity to a CRISPR array are then grouped into clusters of homologous proteins.
  • blastclust is used to form protein clusters.
  • mraseqs2 is used to form protein clusters.
  • a BLAST search of each member of the protein family may be performed over the complete set of known and predicted proteins previously compiled.
  • UBLAST or mmseqs2 may be used to search for similar proteins.
  • a search may be performed only for a representative subset of proteins in the family.
  • the clusters of proteins within close proximity to CRISPR. arrays are ranked or filtered by a metric to determine co-occurrence.
  • One exemplar ⁇ ' metric is the ratio of the size of the protein cluster against the number of BLAST matches up to a certain E value threshold.
  • a constant E value threshold may be used.
  • the E value threshold may be determined by the most distant members of the protein cluster.
  • the global set of proteins is clustered and the cooccurrence metric is the ratio of the size of the CRISPR associated cluster against the size(s) of the containing global cluster(s).
  • a manual review process is used to evaluate the potential functionality and the minimal set of components of an engineered system based on the naturally occurring locus structure of the proteins in the cluster.
  • a graphical representation of the protem cluster may assist in the manual review, and may contain information including pairwise sequence similarity, phylogenetic tree, source organisms / environments, and a graphical depiction of locus structures.
  • the graphical depiction of locus structures may filter for nearby protein families that have a high representation.
  • representation may be calculated by the ratio of the number of related nearby proteins against the size(s) of the containing global cluster(s).
  • the graphical representation of the protein cluster may contain a depiction of the CRISPR array structures of the naturally occurring loci, in some embodiments, the graphical representation of the protein cluster may contain a depiction of the number of conserved direct repeats versus the length of the putative CRISPR array, or the number of unique spacer sequences versus the length of the putative CRISPR array.
  • the graphical representation of the protem cluster may contain a depiction of various metrics of co-occurrence of the putative effector with CRISPR arrays predict new CRiSPR-Cas systems and identify their components.
  • CRISPR-Cas defense systems contains a wide range of activity mechanisms and functional elements that can be harnessed for programmable biotechnologies.
  • these mechanisms and parameters enable efficient defense against foreign DNA and viruses while providing self vs. non-self-discrimination to avoid self-targeting.
  • the same mechanisms and parameters also provide a di verse toolbox of molecular technologies and define the boundaries of the targeting space.
  • systems Cas9 and Casl3a have canonical DNA and RNA endonuclease activity and their targeting spaces are defined by the proiospacer adjacent motif (PAM) on targeted DNA and protospacer flanking sites (PFS) on targeted RNA, respectively.
  • PAM proiospacer adjacent motif
  • PFS protospacer flanking sites
  • the methods described herein can be used to discover additional mechanisms and parameters within single submit Class 2 effector systems that can be more effectively harnessed for programmable biotechnologies.
  • DNA synthesis was used to assemble the components together: in another embodiment, molecular clonmg was used for assembly, in another embodiment, the proteins and noncodmg elements are transcribed on a single mRNA transcript, and different ribosomal binding sites are used to translate individual proteins.
  • spacer direct repeat sequence was cloned into the engineered locus, in one embodiment, the spacers were targeting a second plasmid, pACYC 184, and the spacers were of the length found in the natural CRiSPR array.
  • This crR A library was cloned into the vector backbone containing the proteins and noncoding elements (e.g. pET-28a+), and then subsequently transformed the library into E. coli along with the second target plasmid (e.g., pACYC184). It is important to have the plasmid(s) containing the engineered loci be on compatible origin(s) of replication with respect to the target plasmid to enable bacterial co- transformation. Consequently, each resulting E. coli cell contains no more than one targeting spacer.
  • the E. coli were grown under antibiotic selection.
  • triple antibiotic selection is used: kanamycin for ensuring s uccessful transformation of the pET- 8 ! vector containing the engineered CRISPR-Cas effector system, and chloramphenicol and tetracycline for ensuring successful co-transformation of the pAC YCI 84 target vector.
  • pACYC184 normally confers resistance to chloramphenicol and tetracycline
  • positi ve activity of the novel CRISPR-Cas system targeting the plasmid will eliminate cells that actively express the proteins, noncoding elements, and specific active elements of the crRNA library.
  • deep sequencing e.g., next-generation sequencing
  • mapping the active crRNAs from the pooled screen onto pACYC184 provides patterns of activity that can be suggestive of different activity mechanisms and functional parameters in a broad, hypothesis-agnostic manner. In this way, the features required for reconstituting the novel CRISPR-Cas system in a heterologous prokaryotic species can be more comprehensively tested and studied.
  • the key advantages of the in vivo pooled-screen described herein include: (1) Versatility - engineered locus design allows multiple proteins and/or noncoding elements to be expressed; the library cloning strategy enables both transcriptional directions of the computationally predicted crRNA to be expressed;
  • Sensitivity - pACYC 184 is a low copy plasmid, enabling high sensitivity for CRISPR-Cas activity since even modest interference rates can eliminate the antibiotic resistance encoded by the plasmid;
  • RNA-sequencing and protein expression samples can be directly harvested from the surviving cells in the screen.
  • novel CRISPR-Cas families described herein were evaluated using this in vivo pooled-screen to evaluate their operational elements, mechanisms and parameters, as well as then ability to be active and reprogrammed in an engineered system outside of their natural cellular environment.
  • Example 1 Building an Expanded Database of CRISPR-Cas Systems, and Searching for Type VI-D RNA-Targeting Systems
  • Genome and metagenome sequences were downloaded from NCBI (Benson et al, 2013; Pruitt et al, 2012), NCBI whole genome sequencing (WGS), and DOE JGI Integrated Microbial Genomes (Markowitz et al, 2012). Proteins were predicted (Meta-GeneMark (Zhu et al, 2010) using the standard model MetaGeneMark vl.mod, and Prodigal (Hyatt et al., 2010) in anon mode) on all contigs at least 5kb in length, and de-duplicated in favor of pre-existing annotations to construct a complete protein database.
  • CRISPR arrays were identified and protein sequences for ORFs located within +/- lOkb from CRISPR arrays were grouped into CRISPR-proximal protein clusters. Clusters of fewer than 4 proteins, or comprising proteins from fewer than 3 contigs were discarded. Each of these remaining protein clusters were considered to be a putative effector of a CRISPR-Cas system. In addition to the CRISPR array and putative effector protein, many CRISPR-Cas systems also include additional proteins that enable adaptation, crRNA processing, and defense. Potential additional CRISPR-Cas system components associated with each of the predicted effectors were identified as clusters of protein-coding genes with high effector co-occurrence, and CRISPR enrichment or CRISPR representation of at least 15%.
  • Effector co-occurrence was calculated as the percentage of loci containing the effector that also contain the potential co-occurring protein.
  • the high co-occurrence threshold was a function of the cohesiveness of the effector cluster (more homogenous clusters requiring a higher threshold).
  • the CRISPR enrichment was calculated as follows: 1) Up to 20 unique proteins were sampled from each protein cluster, and UBLAST (Edgar, 2010) was used to generate a rank ordered list of proteins by E-value from the complete protein database, 2) An E-value threshold was imposed to recover at least 50% of the members of the cluster, and 3) CRISPR enrichment was calculated by dividing the number of CRISPR-proximal proteins below the E-value threshold by the total number of proteins below the threshold.
  • CRISPR representation was calculated as the percentage of effector-proximal proteins in a CRISPR- proximal protein cluster. All clustering operations were performed using mmseqs2
  • CRISPR-Cas systems each composed of: 1) a CRISPR array, 2) a putative effector, and optionally, 3) clusters of potential co-acting proteins.
  • a database of (predicted) CRISPR-Cas systems each composed of: 1) a CRISPR array, 2) a putative effector, and optionally, 3) clusters of potential co-acting proteins.
  • Casl3d effectors are notably smaller than previously characterized class
  • Type VI-D loci encode CRISPR-Cas systems that are active in both adaptation and interference.
  • Phylogenetic analysis indicates that Casl proteins associated with Type VI-D are monophyletic and, in accord with previous observations on other type VI systems, are affiliated with the type II-A clade (FIG. 3).
  • the adaptation module seems to have co-evolved with the effector module.
  • Spacer sequences from CRISPR arrays within 3kb of Casl3d effectors were extracted. In the case of multiple contigs containing the same Casl3d sequence (e.g., duplicated locus), only the contig containing the longest CRISPR array was used. Subsequent spacer analysis closely follows the method described previously (Shmakov et al., 2017b). Briefly, the resulting 198 spacers were de-duplicated by comparison of direct and reverse complement sequences, to produce a set of 182 unique spacers.
  • prophage regions To identify prophage regions, (i) all ORFs within 3kb of prokaryotic matches were collected; (ii) a PSI-BLAST search was conducted against the proteins extracted from the virus part of NCBI, using the command line parameters -seg no - evalue 0.000001 -dbsize 20000000; and (iii) a spacer hit was classified as prophage if it overlapped with an ORF with a viral match, or if two or more ORFs with viral matches were identified within the neighborhood of the spacer hit.
  • the CRISPR arrays adjacent to Casl 3d genes contain 198 spacers total, of which 182 are unique.
  • a BLASTN search of the unique spacer sequences against a database comprising known phages and NCBI prokaryotic sequences revealed 7 spacers with significant hits (defined as E-value ⁇ 0.0001, alignment length at least 24, 0 gaps, and no more than one mismatch).
  • a different spacer, from a gut metagenome sequence, resulted in a significant match against a putative prophage region in Bacillus soli (alignment length 24, 0 mismatches).
  • the presence of spacers homologous to DNA phage genomic sequences in an RNA-targeting CRISPR-Cas system might appear unexpected but is in line with similar observations on type VI-A and type VI-B systems (Smargon et al, 2017). Presumably, type VI systems abrogate the reproduction of DNA phages by cleaving phage mRNAs, but the mechanistic details of the antivirus activity of these systems remain to be characterized experimentally.
  • WYL1 is a single WYL-domain protein associated primarily with Ruminococcus .
  • Multiple sequence alignment of WYL 1 shows an N-terminal RHH domain, as well as a pattern of primarily hydrophobic conserved residues, including an invariant tyrosine-leucine doublet corresponding to the original WYL motif (FIG. 7).
  • Other VI-D loci contain duplicated genes encoding WYL-domain proteins, as in Ruminococcus flavefaciens, or a fusion of two WYL-domain proteins, as in Eubacterium sp.
  • VI-D loci encode WYL-domain proteins
  • phylogenetic analysis indicates that these CRISPR-associated WYL proteins are scattered among different branches of the WYL family tree, i.e., are polyphyletic.
  • the VI-D CRISPR-Cas systems appear to have acquired WYL-domain proteins on several independent occasions, suggesting a role for this protein in modulating the CRISPR-Cas function.
  • Exemplary Type VI-D CRISPR-Cas effector proteins are provided in TABLES 1 and 2 (e.g., SEQ ID NOs. 1-31, and 200-350).
  • Exemplary Type VI-D CRISPR-Cas direct repeat sequences are provided in TABLE 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589).
  • Exemplary Type VI-D CRISPR-Cas associated WYL accessory proteins are provided in TABLES 1, 4, 5, and 6.
  • a Type VI-D CRISPR-Cas effector protein comprises an exemplary motif provided in TABLE 7 (e.g., SEQ ID NOs: 94-98, 672 and 673).

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Abstract

L'invention concerne de nouveaux systèmes, procédés et compositions pour la manipulation ciblée d'acides nucléiques. L'invention concerne des systèmes CRISPR génétiquement modifiés, non naturels, des composants et des procédés de modification ciblée d'un acide nucléique.The present invention provides novel systems, methods and compositions for the targeted manipulation of nucleic acids. The present invention relates to genetically modified, non-natural CRISPR systems, components and methods for targeted modification of a nucleic acid.

Description

This application claims the benefit of priority of U.S. Application No. 62/527,957, filed June 30, 2017; U.S. Application No. 62/572,367, filed October 13, 2017; U.S.
Application No. 62/580,880, filed November 2, 2017; U.S. Application No. 62/587,381, filed November 16, 2017; U.S. Application No. 62/619,691, filed January 19, 2018; U.S.
Application No. 62/626,679, filed February 5, 2018; U.S. Application No. 62/628,921, filed February 9, 2018; U.S. Application No. 62/635,443, filed February 26, 2018; U.S.
Application No. 1.5/916,271 , filed March 8, 2018; and U.S. Application No. 15/916,274, filed March 8, 2018. The content of each of the foregoing applications is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present disclosure relates to novel CRISPR systems and components, systems for detecting CRISPR systems, and methods and compositions for use of the CRISPR systems in, for example, nucleic acid targeting and manipulation.
BACKGROUND
Recent advances in genome sequencing technologies and analysis have yielded significant insights into the genetic underpinning of biological acti vities in many diverse areas of nature, ranging from prokaryotic biosynthetic pathways to human pathologies. To fully understand and evaluate the vast quantity of information produced by genetic sequencing technologies, equivalent increases in the scale, efficacy, and ease of technologies for genome and epigenome manipulation are needed. These novel genome and epigenome engineering technologies will accelerate the development of novel applications in numerous areas, including biotechnology, agriculture, and human therapeutics.
Clustered Regularly interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes, collectively known as the CRISPR-Cas or CRISPR/Cas systems, are currently understood to provide immunity to bacteria and archaea against phage infection. The CRISPR-Cas systems of prokaryotic adaptive immunity are an extremely- diverse group of proteins effectors, non-coding elements, as well as loci architectures, some examples of which have been engineered and adapted to produce important biotechnologies.
The components of the systems involved in host defense include one or more effector proteins capable of modifying DNA or RNA and a RNA guide element that is responsible for targeting these protein activities to a specific sequence on the phage DNA or RNA. The RN A guide is composed of a CRISPR RNA (crRNA) and may require an additional trans- activating RNA (tracrRNA) to enable targeted nucleic acid manipulation by the effector protein(s). The crRNA consists of a direct repeat (DR) responsible for protein binding to the crRN A and a spacer sequence, which may be engineered to be complementary to a desired nucleic acid target sequence. In this way, CRISPR systems can be programmed to target DNA or RNA targets by modifying the spacer sequence of the crRNA.
CRISPR-Cas systems can be broadly classified into two classes: Class 1 systems are composed of multiple effector proteins that together form a complex around a crRNA, and Class 2 systems that consist of a single effector protein that complexes with the crRNA to target DNA or RNA substrates. The single-subunit effector compositions of the Class 2 systems provide a simpler component set for engineering and application translation, and has thus far been important sources of programmable effectors. The discovery, engineering, and optimization of novel Class 2 systems may lead to widespread and powerful programmable technologies for genome engineering and beyond.
SUMMARY
CRISPR-Cas systems are adaptive immune systems in archaea and bacteria that defend the species against foreign genetic elements. The characterization and engineering of Class 2 CRISPR-Cas systems, exemplified by CRISPR-Cas9, have paved the way for a diverse array of biotechnology applications in genome editing and beyond. Nevertheless, there remains a need for additional programmable effectors and systems for modifying nucleic acids and polynucleotides (i.e., DNA, RNA, or any hybrid, derivative, or
modification) beyond the current CRISPR-Cas sy stems that enable novel applications through their unique properties.
The present disclosure provides methods for computational identification of new single-effector CRISPR Class 2 sy stems from genomic databases, together with the development of the natural loci into engineered systems, and experimental validation and application translation. In on aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; and ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350), wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence
complementary to the RNA guide spacer sequence.
In some embodiments, the effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the effector protein includes at least two HEPN domains. In some embodiments, none, one, or two or more of the HEPN domains are catalytically deactivated.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR- associated protein or a nucleic acid encoding the CRISPR-associated protein; and iii) an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, wherein the WYL domain includes an amino acid sequence PXXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid; and/or at least one ribbon-ribbon-helix (RHH) fold or at least one helix-tum-helix (HTH) domain; wherein the CRISPR-associated protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR- associated protein or a nucleic acid encoding the CRISPR-associated protein; and an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78- 93, and 590-671); wherein the CRISPR-associated protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
In some embodiments, the activity is a nuclease activity (e.g., a DNAse activity, a targeted RNAse activity, or a collateral RNAse activity).
In some embodiments, the accessory protein increases the activity of the CRISPR- associated protein. In some embodiments, the accessory protein decreases the activity of the CRISPR-associated protein.
In some embodiments, the accessory protein includes or consists of an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein includes or is RspWYLl (SEQ ID NO: 81).
In some embodiments, the targeting of the target nucleic acid results in a modification of the target nucleic acid.
In some embodiments, the CRISPR-associated protein is a Class 2 CRISPR-Cas system protein. In some embodiments, the CRISPR-associated protein includes a RuvC domain (e.g., at least one, two, three, or more RuvC domains). In some embodiments, the CRISPR-associated protein is selected from the group consisting of a Type VI Cas protein, a Type V Cas protein, and a Type II Cas protein. In some embodiments, the CRISPR- associated protein is a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, or a Cas9 protein. In some embodiments, the CRISPR-associated protein is a Type VI-D CRISPR-Cas effector protein comprising at least two HEPN domains, wherein none, one, or two or more of the HEPN domains are catalytically deactivated.
In some embodiments, the effector protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein includes or is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the target nucleic acid is an RNA. In some embodiments, the target nucleic acid is a DNA.
In some embodiments, the modification of the target nucleic acid is a cleavage event. In some embodiments, the modification results in: (a) decreased transcription; (b) decreased translation; or (c) both (a) and (b), of the target nucleic acid. In some embodiments, modification results in (a) increased transcription; (b) increased translation; or (c) both (a) and (b), of the target nucleic acid.
In some embodiments, the effector protein includes one or more amino acid substitutions within at least one of the HEPN domains. In some embodiments, the one or more one amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions result in a reduction of an nuclease activity of the Type VI-D CRISPR-Cas effector protein, as compared to the nuclease activity of the Type VI-D CRISPR-Cas effector protein without the one or more acid substitutions.
In some embodiments, the RNA guide includes a direct repeat sequence that includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs: 32-49, 52-77, 351-589). In some embodiments, the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G. In some embodiments, the direct repeat sequence includes or consists of either 5'-CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
In some embodiments, the spacer includes or consists of from about 15 to about 42 nucleotides. In some embodiments, the RNA guide further includes a trans-activating CRISPR RNA (tracrRNA).
In some embodiments, the systems include a single-stranded donor template or a double-stranded donor template. In some embodiments, the donor template is a DNA or an RNA.
In some embodiments, the systems include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
In some embodiments, the systems are present in a delivery system (e.g., a nanoparticle, a liposome, an adeno-associated virus, an exosome, a microvesicle, and a gene- gun).
In another aspect, the disclosure provides a cell including any of the systems described herein. In some embodiments, the cell is a eukaryotic cell (e.g., a mammalian cell or a plant cell). In some embodiments, the cell is a prokaryotic cell (e.g., a bacterial cell).
In another aspect, the disclosure provides an animal model or a plant model including a cell that includes any of the systems described herein.
In another aspect, the disclosure provides methods of cleaving a target nucleic acid (and compositions for use in such methods), which include contacting a target nucleic acid with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR-associated protein or the Type VI- D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR effector protein cleaves the target nucleic acid. In some embodiments, the target nucleic acid is within a cell.
In another aspect the disclosure provides methods of inducing dormancy or death of a cell which include contacting the cell with a system described herein (and compositions for use in such methods), wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR-associated protein or the Type VI- D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell. In some embodiments, the death is via apoptosis, necrosis, necroptosis, or a combination thereof.
In some embodiments of any of the methods described herein (and compositions for use in such methods), the target nucleic acid is an RNA selected from the group consisting of an mRNA, a tRNA, a ribosomal RNA, a non-coding RNA, a lncRNA, or a nuclear RNA. In some embodiments of any of the methods described herein, the target nucleic acid is a DNA selected from the group consisting of chromosomal DNA, mitochondrial DNA, single- stranded DNA, or plasmid DNA.
In some embodiments of any of the methods described herein (and compositions for use in such methods), upon binding of the complex to the target nucleic acid, the CRISPR- associated protein or the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity.
In some embodiments of any of the methods described herein (and compositions for use in such methods), the cell is a cancer cell (e.g., a tumor cell). In some embodiments, the cell is an infectious agent cell or a cell infected with an infectious agent. In some embodiments, the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
In another aspect, the disclosure provides methods of treating a condition or disease in a subject in need thereof and compositions for use in such methods. The methods include administering to the subject a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject.
In some embodiments of the methods described herein (and compositions for use in such methods), the condition or disease is a cancer or an infectious disease. In some embodiments, the condition or disease is cancer, and wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.
In another aspect, the disclosure provides the use of a system described herein in a method selected from the group consisting of RNA sequence specific interference; RNA sequence-specific gene regulation; screening of RNA, RNA products, IncRNA, non-coding RNA, nuclear RNA, or mRNA; mutagenesis; inhibition of RNA splicing; fluorescence in situ hybridization; breeding; induction of cell dormancy; induction of cell cycle arrest; reduction of cell growth and/or cell proliferation; induction of cell anergy; induction of cell apoptosis; induction of cell necrosis; induction of cell death; or induction of programmed cell death.
In some embodiments of any of the systems described herein, the effector protein is fused to a base-editing domain, an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor. In some embodiments of any of the systems described herein, the CRISPR-associated protein is fused to a base-editing domain (e.g., Adenosine Deaminase Acting on RNA (ADAR) 1 (ADAR1), ADAR2, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)), and activation-induced cytidine deaminase (AID)), an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
In some embodiments, the systems described herein include an RNA-binding fusion polypeptide that includes an RNA-binding domain (e.g., MS2) and a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID).
In another aspect, the disclosure provides method of modifying an RNA molecule, comprising contacting the RNA molecule with a system described herein.
In yet another aspect, the disclosure provides methods of detecting a target RNA in a sample (and compositions for use in such methods). The methods include: a) contacting the sample with: (i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex;
wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single- stranded target RNA in the sample. In some embodiments, the methods further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample. In some embodiments, the target RNA is single-stranded. In some embodiments, the target RNA is double-stranded. In some embodiments, the methods further include transcribing (e.g., using a T7 polymerase) a DNA molecule (e.g., a DNA molecule present in the sample) to produce the target RNA. In some embodiments, the target RNA was transcribed from a DNA molecule. In some embodiments, the methods further include pre-amplifying a nucleic acid in the sample (e.g., via isothermal amplification, recombinase polymerase amplification (RPA), or immunoprecipitation) prior to the contacting step.
In some embodiments, the methods further include contacting the sample with an accessory protein comprising at least one WYL domain. In some embodiments, the accessory protein includes an amino acid sequence having at least 80% (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6. In some embodiments, the accessory protein includes or is RspWYLl (SEQ ID NO: 81).
In some embodiments, the effector protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1 -31 , and 200-350).
In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
In some embodiments, the labeled detector RNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair. In some embodiments, the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein. In some embodiments, a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is decreased. In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is increased.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid; ii) a CRISPR-associated protein or a nucleic acid encoding the CRISPR-associated protein; and an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671); wherein the CRISPR-associated protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence
complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
In some embodiments, the activity is a nuclease activity (e.g., a DNAse activity or an RNAse activity). In some embodiments, the RNAse activity is targeted RNAse activity or a collateral RNAse activity.
In some embodiments, the accessory protein increases the activity of the CRISPR- associated protein. In some embodiments, the accessory protein decreases the activity of the CRISPR-associated protein.
In some embodiments, the accessory protein includes one WYL domain. In some embodiments, the accessory protein includes two WYL domains. In some embodiments, the accessory protein further includes a helix-tum-helix (HTH) fold. In some embodiments, the accessory protein further includes a ribbon-helix-helix (RHH) fold. In some embodiments, the accessory protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590- 671). In some embodiments, the accessory protein includes or consists of an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein is RspWYLl (SEQ ID NO: 81).
In some embodiments, the target nucleic acid includes or is an RNA. In some embodiments, the target nucleic acid includes or is a DNA.
In some embodiments, the targeting of the target nucleic acid results in a modification (e.g., a cleavage event) of the target nucleic acid. In some embodiments, the modification results in cell toxicity. In some embodiments, the modification results in decreased transcription and/or decreased translation of the target nucleic acid. In some embodiments, the modification results in increased transcription and/or increased translation of the target nucleic acid.
In some embodiments, the CRISPR-associated protein is a Class 2 CRISPR-Cas system protein. In some embodiments, the CRISPR-associated protein includes a RuvC domain. In some embodiments, the CRISPR-associated protein is selected from the group consisting of a Type VI Cas protein, a Type V Cas protein, and a Type II Cas protein. In some embodiments, the CRISPR-associated protein is a Cas 13a protein, a Cas 13b protein, a Casl3c protein, a Casl2a protein, or a Cas9 protein.
In some embodiments, the CRISPR-associated protein is a Type VI-D CRISPR-Cas effector protein comprising at least two HEPN domains (e.g., two, three, four, or more HEPN domains). In some embodiments, the Type VI-D CRISPR-Cas effector protein includes two HEPN domains. In some embodiments, at least one (e.g., one, two, three, four, or more) of the HEPN domains is catalytically inactivated.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350). In some embodiments, the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of one or more (e.g., two, three, four, five or six) amino acid substitutions within at least one of the HEPN domains. In some embodiments, the Type VI-D CRISPR-Cas effector protein includes six or less (e.g., five, four, three, two or one) amino acid substitutions within at least one of the HEPN domains. In some embodiments, the one or more one amino acid substitutions include or consist of an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions result in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more acid substitutions.
In some embodiments, the CRISPR-associated proteins include or consist of at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the CRISPR-associated protein include or consist of at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
In some embodiments, the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Χβ is A or G. In some embodiments, the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G. In some embodiments, the direct repeat sequence includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589). In some embodiments, the direct repeat sequence includes or consists of either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
In some embodiments, the spacer includes from about 15 to about 42 nucleotides. In some embodiments, the RNA guide includes a trans-activating CRISPR RNA (tracrRNA). In some embodiments of the systems described herein, the systems include a single- stranded donor template or a double-stranded donor template (e.g., a single-stranded DNA, a double stranded DNA, a single-stranded RNA, or a double stranded RNA).
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3 ' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Xe is A or G; and ii) a Type VI-D CRISPR- Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to and of targeting the target nucleic acid sequence complementary to the RNA guide spacer sequence, and wherein the target nucleic acid is an RNA.
In one aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Xe is A or G; and ii) a Type VI-D CRISPR- Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to and of targeting the target nucleic acid sequence complementary to the RNA guide spacer sequence, and wherein the target nucleic acid is an RNA.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes at least two HEPN domains. In some embodiments, the protein is about 1200 amino acids or less (e.g., 1100, 1000, 1050, 900, 950, 800 amino acids) in length.
In other embodiments, the targeting of the target nucleic acid results in a modification of the target nucleic acid. In some embodiments, the modification of the target nucleic acid is a cleavage event. In some embodiments, the modification results in cell toxicity.
In some embodiments, the modification results in decreased transcription and/or decreased translation of the target nucleic acid. In some embodiments, the modification results in increased transcription and/or increased translation of the target nucleic acid.
In various embodiments, the systems further include a donor template nucleic acid. In some embodiments, the donor template nucleic acid is a DNA or an RNA.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes one or more (e.g., two, three, four, five or six) amino acid substitutions within at least one of the HEPN domains. In some embodiments, the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2. In some embodiments, the one or more amino acid substitutions result in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more amino acid substitutions.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350). In some embodiments, the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the systems include an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein includes at least one WYL domain, and wherein the accessory protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein includes two WYL domains. In some embodiments, the accessory protein further includes a helix-tum-helix (HTH) fold and/or a ribbon-helix-helix (RHH) fold. In some embodiments, the accessory protein is RspWYLl (SEQ ID NO: 81). In some embodiments, the accessory protein modulates (e.g., increases or decreases) an activity of the Type VI-D CRISPR-Cas effector protein. In some embodiments, the activity is an RNAse activity, an RNA-binding activity, or both. In some embodiments, the RNAse activity is a targeted RNAse activity or a collateral RNAse activity.
In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the CRISPR-associated protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.
In some embodiments, the direct repeat sequence includes or consists of a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589). In some embodiments, the direct repeat sequence includes or consists of either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
In some embodiments, the spacer sequence includes or consists of from about 15 to about 42 nucleotides.
In some embodiments, the systems provided herein include a single-stranded donor template or a double-stranded donor template (e.g., an RNA or a DNA molecule).
In some embodiments, the systems provided herein include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Χβ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein and/or a nucleic acid encoding the effector protein, wherein the effector protein is about 1200 or fewer amino acids, and wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein and/or a nucleic acid encoding the effector protein, wherein the effector protein is about 1200 or fewer amino acids, and wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Χβ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is about 950 or fewer amino acids in length, and wherein the effector protein is capable of binding (e.g., binds under appropriate conditions), to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G; and ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is about 950 or fewer amino acids in length, and wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of : i) an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Χβ is A or G; ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence; and iii) an accessory protein, wherein the accessory protein includes at least one WYL domain, wherein the accessory protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671), and wherein the accessory protein is capable of regulating (e.g., regulates under appropriate conditions) an activity of the effector protein.
In another aspect, the disclosure provides engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: i) an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G; ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence; and iii) an accessory protein, wherein the accessory protein includes at least one WYL domain, wherein the accessory protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6 (e.g., SEQ ID NOs. 78-93, and 590-671), and wherein the accessory protein is capable of regulating (e.g., regulates under appropriate conditions) an activity of the effector protein.
In some embodiments, the accessory protein is RspWYLl (SEQ ID NO: 81).
In some embodiments, the effector protein includes at least two HEPN domains. In some embodiments, the effector protein includes or consists of an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the CRISPR-associated protein (e.g., Type VI-D CRISPR-Cas effector protein) is fused to a base-editing domain (e.g., Adenosine Deaminase Acting on RNA (ADAR) 1; ADAR2; apolipoprotein B mRNA editing enzyme, catalytic polypepti de- like (APOBEC); and activation-induced cytidine deaminase (AID)). In some embodiments, the base-editing domain is further fused to an RNA-binding domain.
In some embodiments, the CRISPR associated protein (e.g., a Type VI-D CRISPR- Cas effector protein) is fused to a RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
In some embodiments, the CRISPR-associated (e.g., a Type VI-D CRISPR-Cas effector protein) further includes a linker sequence. In some embodiments, the CRISPR- associated protein (e.g., a Type VI-D CRISPR-Cas effector protein) includes one or more mutations or amino acid substitutions that render the CRISPR-associated protein unable to cleave RNA.
In some embodiments, the systems described herein also include an RNA-binding fusion polypeptide that includes an RNA-binding domain and a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, and AID). In some embodiments, the RNA-binding domain is MS2, PP7, or Qbeta.
In some embodiments, the systems described herein include a nucleic acid encoding the CRISPR-associated protein (e.g., a Type VI-D CRISPR-Cas effector protein). In some embodiments, the nucleic acid encoding the CRISPR-associated protein is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the nucleic acid encoding the CRISPR-associated protein is codon-optimized for expression in a cell (e.g., a mammalian cell or a bacterial cell).
In some embodiments, the systems described herein include a nucleic acid encoding the accessory protein. In some embodiments, the nucleic acid encoding the accessory protein is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the nucleic acid encoding the accessory protein is codon-optimized for expression in a cell.
In some embodiments, the systems described herein include a nucleic acid encoding one or more RNA guides (e.g., crRNAs). In some embodiments, the nucleic acid encoding the one or more RNA guides is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
In some embodiments, the systems described herein include a nucleic acid encoding a target nucleic acid (e.g., a target RNA). In some embodiments, the nucleic acid encoding the target nucleic acid is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).
In some embodiments, the systems described herein include a nucleic acid encoding a CRISPR-associated protein and a nucleic acid encoding an accessory protein in a vector. In some embodiments, the system further includes one or more nucleic acids encoding an RNA guide present in the vector.
In some embodiments, the systems provided herein include a nucleic acid encoding a Type VI-D CRISPR-Cas effector protein in a vector.
In some embodiments, the systems provided herein include a nucleic acid encoding the Type VI-D CRISPR-Cas effector protein and a nucleic acid encoding the accessory protein in a vector. In some embodiments, the system further includes one or more nucleic acids encoding one or more RNA guides (e.g., crRNAs) in the vector.
In some embodiments, the vectors included in the systems are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors. In some embodiments, the vectors included in the system are phage vectors.
In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microvesicle, and a gene-gun.
The disclosure also provides a cell (e.g., a eukaryotic cell or a prokaryotice cell (e.g., a bacterial cell)) comprising a system described herein. In some embodiments, the eukaryotic cell is a mammalian cell (e.g., a human cell) or a plant cell. The disclosure also provides animal models (e.g., rodent, rabbit, dog, monkey, or ape models) and plant model that include the cells.
In another aspect, the disclosure provides methods of cleaving a target nucleic acid (and compositions for use in such methods), wherein the methods include contacting the target nucleic acid with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the CRISPR- associated protein or the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR effector protein cleaves the target nucleic acid. In some
embodiments of the methods, the target nucleic acid is within a cell.
In another aspect, the disclosure also provides methods of inducing dormancy or death of a cell (and compositions for use in such methods), wherein the methods include contacting the cell with a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid, wherein the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell. In some embodiments of the methods described herein, the death of the cell is via apoptosis, necrosis, necroptosis, or a combination thereof.
In some embodiments, the target nucleic acid is an RNA molecule (e.g., an mRNA, a tRNA, a ribosomal RNA, a non-coding RNA, a IncRNA, or a nuclear RNA). In some embodiments, the target nucleic acid is a DNA molecule (e.g., chromosomal DNA, mitochondrial DNA, single-stranded DNA, or plasmid DNA).
In some embodiments of the methods described herein, upon binding of the complex to the target nucleic acid, the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity.
In some embodiments, the cell is a cancer cell (e.g., a tumor cell). In some embodiments, the cell is an infectious agent cell or a cell infected with an infectious agent. In some embodiments, the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
In another aspect, the disclosure provides methods of treating a condition or disease in a subject in need thereof (and compositions for use in such methods, the methods include administering to the subject a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject.
In some embodiments, the condition or disease is a cancer or an infectious disease. In some embodiments, the condition or disease is cancer, and wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.
In another aspect, the disclosure provides the use of a system described herein in a method selected from the group consisting of RNA sequence specific interference; RNA sequence-specific gene regulation; screening of RNA, RNA products, IncRNA, non-coding RNA, nuclear RNA, or mRNA; mutagenesis; inhibition of RNA splicing; fluorescence in situ hybridization; breeding; induction of cell dormancy; induction of cell cycle arrest; reduction of cell growth and/or cell proliferation; induction of cell anergy; induction of cell apoptosis; induction of cell necrosis; induction of cell death; or induction of programmed cell death.
In some embodiments, the methods described herein are performed either in vitro, in vivo, or ex vivo.
The disclosure also provides methods of modifying an RNA molecule (and compostions for use in such methods), including contacting the RNA molecule with a system described herein. In some embodiments, the spacer sequence is complementary to at least 15 nucleotides of the RNA molecule.
The disclosure also provides methods of detecting a target RNA (e.g., a single- stranded RNA or a double-stranded RNA) in a sample, the methods including: a) contacting the sample with: (i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR- Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single- stranded target RNA in the sample.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes at least two HEPN domains. In some embodiments, the Type VI-D CRISPR-Cas effector protein is about 1200 amino acids or less in length.
In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, the Type VI-D CRISPR-Cas effector protein includes or consists of an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350). In some embodiments, the Type VI-D CRISPR-Cas effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
In some embodiments, the effector protein includes one or more amino acid substitutions within at least one of the HEPN domains. In some embodiments, the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
In some embodiments, the methods further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
In some embodiments, the labeled detector RNA includes a fluorescence-emitting dye pair. In some embodiments, the labeled detector RNA includes a fluorescence resonance energy transfer (FRET) pair. In some embodiments, the labeled detector RNA includes a quencher/fluor pair.
In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is decreased. In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is increased. In some embodiments, the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.
In some embodiments, a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
In some embodiments, the labeled detector RNA includes a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof.
In one aspect, the disclosure relates to engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5'- X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein XI is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and X6 is A or G; and a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the target nucleic acid is an RNA.
In one aspect, the disclosure relates to engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) systems that include or consist of: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence includes 5 '- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Xe is A or G; and a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding (e.g., binds under appropriate conditions) to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the target nucleic acid is an RNA.
In some embodiments of these systems, the Type VI-D CRISPR-Cas effector proteins include at least two HEPN domains. In some embodiments, the Type VI-D CRISPR-Cas effector proteins include an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 1 , and SEQ ID NO: 10. In other embodiments, the Type VI-D CRISPR-Cas effector proteins include an amino acid sequence having at least 95% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1 -31 , and 200-350), or they can include an amino acid sequence provided in Table 2.
In various embodiments, the direct repeat sequence can include a nucleotide sequence provided in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589).
In some embodiments, the targeting of the target nucleic acid results in a modification of the target nucleic acid. For example, the modification of the target nucleic acid can be a cleavage event.
In the new systems, the Type VI-D CRISPR-Cas effector proteins can include one or more amino acid substitutions within at least one of the HEPN domains resulting in a reduction of an RNAse activity of the Type VI-D CRISPR-Cas effector protein, as compared to the RNAse activity of the Type VI-D CRISPR-Cas effector protein without the one or more amino acid substitutions, e.g., 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions. In some embodiments, the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
In some embodiments, the Type VI-D CRISPR-Cas effector protein is fused to a base- editing domain, e.g., to an RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
In various embodiments, the Type VI-D CRISPR-Cas effector protein includes at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or both. In some embodiments, the direct repeat sequence includes either 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153). In some embodiments, the spacer consists of from about 15 to about 42 nucleotides.
In another aspect of the disclosure, the systems include the nucleic acid encoding the Type VI-D CRISPR-Cas effector protein, operably linked to a promoter. For example, the promoter can be a constitutive promoter.
In some embodiments, the nucleic acid encoding the Type VI-D CRISPR-Cas effector protein is codon-optimized for expression in a cell. In various embodiments, the nucleic acids encoding the Type VI-D CRISPR-Cas effector protein are operably linked to a promoter within in a vector, e.g., selected from the group consisting of a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector.
In another aspect, the system is present in a delivery system selected from the group consisting of a nanoparticle, a liposome, an exosome, a microvesicle, and a gene-gun.
In some embodiments, the systems can further include a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA includes a sequence that is capable of hybridizing (e.g., hybridizes under appropriate conditions) to the spacer sequence of the RNA guide.
In another aspect, the disclosure includes one or more cells that include the systems described herein.
In another aspect, the disclosure provides methods of cleaving a target nucleic acid. The methods include contacting the target nucleic acid with a system as described herein; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid; wherein the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex;
wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence, the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid.
In another aspect, the disclosure provides methods of inducing dormancy or death of a cell, e.g., in vitro or in vivo (and compositions for use in such methods), the method including contacting the cell with a system as described herein; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid within the cell; wherein the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex; wherein the complex binds to the target nucleic acid sequence that is
complementary to the at least 15 nucleotides of the spacer sequence; and wherein after binding of the complex to the target nucleic acid sequence, the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell.
In these methods, the cell can be a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
In other embodiments, the disclosure provides methods of modifying a target nucleic acid in a sample, in which the methods include contacting the sample with a system as described herein, e.g., with fusion proteins; wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid within the sample; wherein the Type VI-D CRISPR-Cas effector protein fused to the base editing domain associates with the RNA guide to form a complex; wherein the complex binds to the target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein after binding of the complex to the target nucleic acid sequence, the Type VI-D CRISPR-Cas effector protein fused to the base-editing domain modifies at least one nucleobase of the target nucleic acid. In another aspect, the disclosure provides methods of detecting a single-stranded target RNA in a sample. These methods include: a) contacting the sample with: (i) a RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single-stranded target RNA in the sample.
In these methods, the effector protein includes an amino acid sequence having at least 90% sequence identity to an amino acid sequence provided in Table 2 (e.g., SEQ ID NOs. 1- 31, and 200-350). These methods can further include comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
The term "cleavage event," as used herein, refers to a break in a target nucleic acid created by a nuclease (e.g., a Type VI-D CRISPR-Cas effector protein) of a CRISPR. system described herein. In some embodiments, the cleavage event is a single-stranded RNA break. In some embodiments, the cleavage event is a double-stranded RNA break. In some embodiments, the cleavage event is a double-stranded DNA break. In some embodiments, the cleavage event is a single-stranded DNA break.
The terms "CRISPR system" or '"Clustered Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) system" as used herein refer to nucleic acids and/or proteins involved in the expression of, or directing the activity of, CRISPR-efFectors, including sequences encoding CRISPR effectors, RNA guides, and other sequences and transcripts from a CRISPR locus. In some embodiments, the CRISPR system is an engineered, non- naturally occurring CRISPR system In some embodiments, the components of a CRISPR system may include a nucleic acid(s) (e.g., a vector) encoding one or more components of the sy stem, a component(s) in protein form, or a combination thereof.
The term "CRISPR array" as used herein refers to the nucleic acid (e.g., DNA) segment that includes CRISPR repeats and spacers, starting with the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats. The terms "CRISPR repeat/' or "CRISPR direct repeat," or "direct repeat," as used herein, refer to multiple short direct repeating sequences, which show very little or no sequence variation within a CRISPR array.
The term "CRISPR RNA" or "crRNA" as used herein refers to a RNA molecule including a guide sequence used by a CRISPR effector to target a specific nucleic acid sequence. Typically, crRNAs contains a sequence that mediates target recognition and a sequence that forms a duplex with a tracrRNA. In some embodiments, the crRNA: tracrRNA duplex binds to a CRISPR effector.
The terms "donor template" or "donor template nucleic acid," as used herein refers to a nucleic acid molecule that can be used by one or more cellular proteins to modify the sequence of a target nucleic acid after a CRISPR-associated protein described herein has altered the target nucleic acid. In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid. In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear. In some embodiments, the donor template nucleic acid is circular (e.g., a plasraid). In some embodiments, the donor template nucleic acid is an exogenous nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome). In some embodiments, the donor template is a DNA molecule. In some embodiments, the donor template is an RNA molecule.
The term "CRISPR effector," "effector," "CRISPR-associated protein," or "CRISPR enzyme" as used herein refers to a protein that carries out an enzymatic activity or that binds to a target site on a nucleic acid specified by a RNA guide. In different embodiments, a CRISP effector has endonuclease activity, nickase activity, exonuciease activity, transposase activity, and/or excision activity. In some embodiments, the CRISPR-associated protein is a Type VI Cas protein, a Type V Cas protein, or a Type II Cas protein. In some embodiments, the CRISPR-associated protein is a Casl 3a protein, a Cas 13b protein, a Casl3c protein, a Casl3d protein, a Casl2a protein, or a Cas9 protein. In some
embodiments, the CRISPR-associated protein is a Type Vl-D CRISPR-Cas effector protein described herein.
The term "RNA guide" as used herein refers to any RNA molecule that facilitates the targeting of a protein described herein to a target nucleic acid. Exemplary "RNA guides" include, but are not limited to, crRNAs or crR As in combination with cognate trans- activating RNAs (tracrR As). The latter may be independent RNAs or fused as a single RNA using a linker. In some embodiments, the RNA guide is engineered to include a chemical or biochemical modification, in some embodiments, an RNA guide may include one or more nucleotides.
The term "origin of replication," as used herein, refers to a nucleic acid sequence in a replicating nucleic acid molecule (e.g., a plasmid or a chromosome) that is recognized by a replication initiation factor or a DNA replicase.
As used herein, the term "targeting" refers to the ability of a complex including a CRISPR-associ ated protein and a RN A guide, such as a crRNA, to bind to a specific target nucleic acid and not to other nucleic acids that do not have the same sequence as the target nucleic acid.
As used herein, the term "target nucleic acid" refers to a specific nucleic acid sequence that specifically binds to a complex including a CRlSPR-associated protein and a RNA guide described herein. In some embodiments, the target nucleic acid is or includes a gene. In some embodiments, the target nucleic acid is or includes a non-coding region (e.g. , a promoter). In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.
The terms "trans-activating crRNA" or "tracrRNA" as used herein refer to an RNA including a sequence that forms a structure required for a CRlSPR-associated protein to bind to a specified target nucleic acid.
The term "collateral RNAse activity," as used herein in reference to a CRlSPR- associated protein, refers to non-specific RNAse activity of a CRlSPR-associated protein after the enzyme has bound to and/or modified a specifically-targeted nucleic acid. In some embodiments, a CRlSPR-associated protem (e.g., a Type VI-D CRISPR-Cas effector protem) exhibits collateral RNAse activity after binding to a target nucleic acid (e.g., a target RNA). A nucleic acid that is cleaved or degraded by a CRlSPR-associated protein in a non-specific manner (i.e., when the protem exhibits collateral RNAse activity) is referred to herein as a "non-target nucleic acid."
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, in case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
Other features and adv antages of the invention will be apparent from the following detailed description, and from the claims.
BRIEF FIGURE DESCRIPTION
FIG. 1 depicts a schematic representation of a maximum likelihood tree topology for an exemplary subset of Casl3d, with the genomic arrangement of the genes encoding predicted protein components of Type VI-D system components shown to the right. Each locus sequence is identified by a protein accession or gene number, with the species name provided where available. Key proteins and CRISPR arrays are depicted as follows: white - Casl3d, horizontal stripes - WYL1 accessory protein, light gray - WYL domain containing protein, vertical stripes - Casl, dark gray - Cas2.
FIG. 2A depicts a schematic tree comparing the different type VI subtype locus structures. Gene arrows are shown roughly proportional to size. Labels denote the following: WYL - WYL domain, HEPN - HEPN nuclease domain.
FIG. IB depicts a size comparison for Casl3 proteins from the 4 type VI subtypes; error bars specify the mean and standard deviation.
FIG. 3 depicts a phylogenetic tree of Casl proteins from type II and type VI CRISPR- Cas systems. The tree was constructed for a non-redundant set of Casl proteins associated with Casl 3d and type II and type VI CRISPR-Cas systems as described previously (see (Peters et al, 2017)). Several Casl proteins associated with subtype I-E systems were selected for an outgroup. Each sequence is denoted by a local numeric identifier, CRISPR- Cas type and species name (if available). Casl proteins associated with Casl3d are denoted by "CAS-VI-D", and those associated with Casl3a by "CAS-VI-A". Several branches were collapsed and are shown by triangles with CRISPR-Cas system indicated on the right.
Support values are indicated for selected branches. FIGs. 4A and 4B depict a phylogenetic tree constructed for a combined set of Casl3d sequences described (light gray) and previously described Casl3a sequences. Each sequence is denoted by a protein locus tag and species name (if available). Casl3d proteins form a clade with a 100% bootstrap support value (shown on branch).
FIGs. 5A, 5B and5C depict a multiple sequence alignment of Casl3d protein sequences (RspCasl3d (SEQ ID NO: 2) and EsCasl3d (SEQ ID NO: 1) and Casl3a protein sequences (LbaCasl3a (SEQ ID NO: 156), LbuCasl3a (SEQ ID NO: 157), LshCasl3a (SEQ ID NO: 158)). Previously identified domains of Cas 13a are highlighted with varying background shading as indicated in the figure (NTD, N-terminal domain). Note the nearly complete absence of a counterpart to the Helical-1 domain of Casl3a in Casl3d (the alignment in this region cannot be considered reliable).
FIG. 6 depicts a phylogenetic tree of the WYLl protein family. Exemplary WYLl proteins associated with Casl3d are denoted by gray. In cases when a CRISPR array and/or other cas genes are present in the vicinity of the respective WYLl gene (within 10 kb up- and downstream), the description includes "CRISPR". Several branches were collapsed and are indicated by triangles. Domain organization is schematically shown next to each branch. Abbreviation: WYL - WYL domain (usually fused to a characteristic C-terminal
subdomain); RHH - ribbon helix helix superfamily DNA binding domain.
FIG. 7 depicts a multiple sequence alignment of exemplary WYLl protein sequences. The RHH domain is denoted by 'r' and the WYL domain fused to the characteristic C- terminal subdomain is denoted by 'y' underneath the alignment. The predicted secondary structure elements are shown (E, extended conformation (β-strand), H, oc-helix).
FIG. 8 depicts a design of minimal engineered CRISPR-Cas systems for the Rsp and Es type VI-D CRISPR loci (referred to as RspCasl3d and EsCasl3d systems), with a spacer library tiling pACYC184 (both top strand and bottom strand).
FIG. 9 depicts a schematic of the bacterial negative selection screen used to evaluate functional parameters of RspCasl3d and EsCasl3d systems.
FIGs. 10A and 10B depict a negative control condition from bacterial screens for EsCasl3d and RspCasl3d systems, respectively. Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library. Non-targeting CRISPR arrays (with spacers matching a GFP open reading frame) inserted into EsCasl3d and RspCasl3d screening systems showed minimal levels of depletion in bacterial negative selection screens (no GFP open reading frame was included in our screen system).
FIGs. 11A and 11B depict a negative control condition from bacterial screens for EsCasl3d and RspCasl3d systems, respectively. Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library. Deletion of EsCasl3d and RspCasl3d-RspWYLl open reading frames from the EsCasl3d and
RspCasl3d screening systems resulted in minimal depletion of library CRISPR array elements in bacterial negative selection screens.
FIGs. 12A and 12B depict the distribution and magnitude of crRNA depletion from bacterial screens for EsCasl3d and RspCasl3d, respectively. Depletion value was calculated by normalized sequencing reads from the screen output divided by normalized reads from the pre-transformation screen input library for each crRNA spacer and orientation. Solid and dashed lines represent both possible direct repeat (DR) orientations cloned into the screening library, cloned into the screening library. The vertical dashed lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
FIGs. 13A and 13B depict the location of strongly depleted targets of the active DR orientation over the strands and genetic features of the pACYC184 plasmid for EsCasl3d and RspCasl3d systems, respectively. Light gray outlines represent the total number of spacers (y-axis) targeting a location, while short bars depict the locations of strongly depleted spacers with heatmap color proportional to magnitude of depletion. Directional expression data for pACYC184 is plotted as a heatmap between the x-axes.
FIGs 14A and 14B depict web logos for the 5' and 3' 30 nt regions flanking strongly depleted targets for EsCasl3d and RspCasl3d systems, and show no evidence of PFS or PAM requirements.
FIG. 14C depicts violin plots of bit scores of all possible PFS targeting rules of up to length 3 involving the target site and +/- 15 nt flanking region, for BzCasl3b, RspCasl3d, and EsCasl3d systems. Dots represent data points outside of the discernable density of the violin plot. These dots accurately recapitulate the known PFS positions of BzCasl3b, as shown above the dots.
FIG. 15 depicts bar charts showing the fraction of hits for RspCasl3d and EsCasl3d systems according to features of the plasmid for all targets.
FIGs. 16A and 16B depict heatmaps of the fraction (# strongly depleted spacers) / (# strongly depleted spacers + # non-depleted spacers) for all target regions (CRISPR arrays with active direct repeat orientation only) with no predicted secondary structure between specific start (x-axis) and end (y-axis) locations. White boxes indicate specific target regions (bounded by start (x-axis) and end (y-axis) locations), where selection of spacers with no predicted secondary structure maximized targeting efficacy, while minimizing the number of screen spacers eliminated due to the presence of predicted secondary structure. Targets these spacer populations are referred to as "low secondary structure targets" for RspCasl3d and EsCasl3d respectively.
FIG. 16C depicts bar charts showing the fraction of hits for RspCasl3d and
EsCasl3d systems according to features of the plasmid for low secondary structure targets.
FIG. 17 depicts a schematic of the RNA extraction from bacterial screen, next- generation sequencing (NGS), and alignment to determine the mature crRNA for EsCasl3d. Distribution of read counts by crRNA sequence location is depicted on the right, and the predicted EsCasl3d mature crRNA secondary structure is shown.
FIG. 18 depicts a coomassie blue stained polyacrylamide gel of purified recombinant proteins EsCasl3d, RspCasl3d, and RspWYLl respectively.
FIG. 19 depicts schematic representions of the major products identified from next- generation sequencing of in vitro cleaved RNA fragments from the pre-crRNA processing with EsCasl3d and RspCasl3d. The black line represents the direct repeats and associated secondary structure, the box represents the full-length spacer, and the filled triangle represents the cleavage sites. The lengths described are for processed EsCasl3d crRNAs, with RspCasl3d having one extra nucleotide due to the 31nt natural length spacer used for instead of 30. Not depicted are the 3-4 nt at the 5' end of the pre-crRNA from T7 in vitro transcription.
FIGs. 20A, 20B, 20C, and 20D depict denaturing gels displaying Casl3d mediated cleavage of their cognate pre-crRNAs over a dose titration of effector concentration. The dependence of Casl3d crRNA biogenesis on divalent metal cations was evaluated with the introduction of lOOmM EDTA to the standard reaction conditions.
FIG. 21 depicts a denaturing gel displaying LwaCasl3a at a final concentration of lOOnM processing of pre-crRNA (200nM) without the presence of EDTA, and under reaction conditions supplemented with increasing concentrations of EDTA (3.3 - lOOmM).
FIGs. 22A and 22B depict a titration of Apo EsCasl3d and RspCasl3d (100 - 0.4nM) over a non-targeted ssDNA substrate (ΙΟΟηΜ).
FIGs. 23A and 23B depict a titration of EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over non-targeted ssDNA substrates (ΙΟΟηΜ).
FIGs. 24A and 24B depict a titration of EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over targeted ssDNA substrates (ΙΟΟηΜ). Saturation of target cleavage activity was observed at approx. 50nM RspCasl3d-crRNA complex and ΙΟΟηΜ EsCasl3d- crRNA complex.
FIGs. 25A and 25B depict representative denaturing gels displaying the targeted RNase activity of EsCasl3d and RspCasl3d effector proteins, with substrate RNA cleavage occurring when the crRNA matches its complementary target ssRNA. RNA substrates are 5' labeled with IRDye 800.
FIGs. 26A and 26B depict representative denaturing gels displaying non-specific RNase activity of the Casl3d effectors upon targeted substrate recognition, demonstrated by the cleavage of fluorescein dUTP body -labeled collateral RNA upon activation of the target nuclease activity. For all reactions, EsCasl3d-crRNA and RspCasl3d-crRNA complexes were formed by pre-incubating Casl3d and cognate crRNA for 5 minutes at 37°C, prior to adding target and/or collateral ssRNA and incubating the reaction for 30 minutes.
FIGs. 26C and 26D depict denaturing gels displaying cleavage reactions of the Casl3d-crRNA complex over two distinct ssRNA substrates, short 150nt target RNAs (top) and longer 800nt fluorescent body-labeled ssRNA substrates (bottom) for EsCasl3d and RspCasl3d. The labels A and B correspond to matching crRNA / substrate pairs.
FIG. 27A depicts a comparative depletion plot of bacterial screens performed on RspCasl3d only (solid line, long dashes) versus RspCasl3d with RspWYLl (short and medium dashes). The dashed vertical lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
FIG. 27B depicts spacer depletion ratios for RspCasl3d with and without RspWYLl.
FIG. 28 depicts a depletion plot of bacterial screens using only RspWYLl and the repeat-spacer-repeat library associated with RspCasl3d.
FIG. 29A and 29B depict representative activity of titrating different molar ratios of purified RspWYLl to a fixed dose of RspCasl3d. FIG. 29A is an ssRNA substrate cleavage assay, and FIG. 29B evaluate the effect of RspWYLl on collateral activity.
FIG. 29C depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of Apo RspCasl3d (200nM) for target ssRNA.
FIG. 29D depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of Apo RspCasl3d (200nM) for collateral ssRNA activity.
FIG. 29E depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of RspCasl3d-crRNA complex (50nM) for target ssRNA.
FIG. 29F depicts the effect on RNA cleavage of titrating RspWYLl (800 to 0.4nM) while holding fixed the concentration of RspCasl3d-crRNA complex (50nM) for collateral ssRNA activity.
FIGs. 30A and 30B depict representative activity of titrating different molar ratios of purified RspWYLl to a fixed dose of EsCasl3d. FIG. 30A is an ssRNA substrate cleavage assay, and FIG. 30B evaluate the effect of RspWYLl on collateral activity of EsCasl3d. In both of these reactions, RspWYLl was pre-incubated along with the pre-crRNA and Casl3d effector for 5 minutes at 37°C before incubation with substrate RNA. The final concentration of Casl3d in the reaction is 33nM with a 2: 1 ratio of Casl3d to pre-crRNA.
FIG. 31 shows that RspWYLl enhances the activity of type VI-B effector BzCasl3b. Representative gel displaying the ability of RspWYLl to enhance target cleavage and collateral activity for Casl3 enzymes of subtype VI-B, demonstrating modularity beyond Type VI-D. In this reaction RspWYLl was pre-incubated along with the pre-crRNA and BzCasl3b effector for 5 minutes at 37C before incubation with substrate RNA.
FIGs. 32A and 32B show that EsCasl3d and RspCasl Sd, respectively, are capable of specific detection of RNA species using the collateral effect of the enzymes, and additionally, demonstrate differential activity over short ribonucleotide oligomer substrates. The poly-G and poly-U labels refer to substrates containing 5 identical ribonucleotide bases, with the 5' end modified with a FAM labeled fluorescent nbonucleotide and the 3' end modified with an Iowa Black FQ fluorescent quencher. These data were collected 60 minutes after incubation at 37 CC. The error bars represent S.E.M. of four technical replicates.
FIGs. 33A and 333B depict the distribution and magnitude of crRNA depletion for primary screening of EsCasl3d and RspCasl 3d (effector only), respectively, in the absence of tetracycline. The value of crRNA depletion was calculated by normalized sequencing reads from the screen output divided by normalized reads from the pre-transformation screen input library for each crRNA spacer and orientation. The vertical dashed lines demarcate the intersection of the ranked screen hits with the depletion fraction of 0.1, below which we define as strongly depleted.
FIGs. 34A and 34B depict the location of strongly depleted targets of the active DR orientation over the strands and genetic features of the pACYC i84 plasnnd for EsCasBd and RspCaslSd (effector only), respectively. Light gray outlines represent the total number of spacers (y-axis) targeting a location, while short horizontal bars depict the locations of strongly depleted spacers with heatmap color proportional to magnitude of depletion.
DETAILED DESCRIPTION CRISPR Class 2 RNA-Guided RNases
In one aspect, provided herein is a novel family of CRISPR Class 2 effectors having two strictly conserved RX4-6H motifs, characteristic of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains. CRISPR Class 2 effectors that contain two HEPN domains have been previously characterized and include, for example, CRISPR Casl3a (C2c2), Casl3b, and Casl3c.
HEPN domains have been shown to be RNAse domains and confer the ability bind to and cleave any target RNA molecule. In some embodiments, a HEPN domain comprises the amino acid sequence RXXXXH, wherein X is any amino acid (SEQ ID NO: 94). The target RNA may be any suitable form of RNA, including but not limited to mRNA, tRNA, ribosomal RNA, non-coding RNA, lincRNA, and nuclear RNA. For example, in some embodiments, the CRISPR-associated protein recognizes and cleaves targets located on the coding strand of open reading frames (ORFs).
In one embodiment, the disclosure provides a family of CRISPR Class 2 effectors, referred to herein generally as Type VI-D CRISPR-Cas effector proteins, Casl3d or CasBe. Direct comparison of the Type VI-D CRISPR-Cas effector proteins with the effector of these other systems shows that Type VI-D CRISPR-Cas effector proteins are significantly smaller (e.g., 20% fewer amino acids), and have less than 10% sequence similarity in multiple sequence alignments to other previously described effector proteins. This newly-identified family of CRISPR Class 2 effectors can be used in a variety of applications, and are particularly suitable for therapeutic applications since they are significantly smaller than other effectors (e.g., CRISPR Casl3a, Casl3b, or Casl3c effectors) which allows for the packaging of the effectors and/or nucleic acids encoding the effectors into delivery systems having size limitations.
In bacteria, the Type VI-D CRISPR-Cas systems include a single effector
(approximately 920 amino acids in length), and one or none accessory proteins
(approximately 380 amino acids in length) within close proximity to a CRISPR array. The CRISPR array includes direct repeat sequences typically 36 nucleotides in length, which are generally well conserved, especially on the 3' end which ends with TNTNAAAC (SEQ ID NO: 154). Reduced consensus of the nucleotide sequence in the 5' end of the direct repeats suggests that the crRNA is processed from the 5' end. With few exceptions, the 21 nucleotide sequence immediately upstream of the 3' end TNTNAAAC (SEQ ID NO: 154) starts with a highly conserved A and exhibits sequence complementarity that suggests strong base pairing for an RNA loop structure. The spacers contained in the Casl3d CRISPR arrays are most commonly 30 nucleotides in length, with the majority of variation in length contained in the range of 28 to 36 nucleotides.
Exemplary Type VI-D CRISPR-Cas effector proteinsare provided below in Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, a Type VI-D CRISPR-Cas effector proteinsinclude an amino acid sequence having at least about 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of any one of Table 2 (e.g., SEQ ID NOs. 1-31, and 200-350). In some embodiments, a Type VI-D CRISPR-Cas effector proteins includes the amino acid sequence of any one of Table 2 (e.g., SEQ ID NOs. 1-31, and 200- 350). In some embodiments, the Type VI-D CRISPR-Cas effector proteins is DS499551 (SEQ ID NO: 1 ; also referred to herein as EsCasl3d) or LARF01000048 (SEQ ID NO: 2; also referred to herein as RspCasl3d), the amino acid sequences of each are provided below:
>WP_005358205.1 (EsCasl3d)
[ Eubacterium siraeum DSM 15702]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVPKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFG RGNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGRKKDEPEQSVPTDMLCLKPTLEKK FFGKEFDDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADE IENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEK FIGNYRLAYFADAFYVNKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPV EEINNRFIENNKVNIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSI KKLRESMLEGKGYADKEYDSVRNKLYQMTDFIL YTGYINEDSDRADDLVNTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKKLSKSNIEIQEDKLRKCFISYAD SVSEFTKLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDIN AKRTMYRDALDILGIESDKTEEDIEKMIDNILQIDA GDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPA VRFVLNEIPDAQIERYYEACCPKNTALCSA KRREKLADMIAEIKFENFSDAGNYQKA VTSRTSEAEIKRKNQAIIRLYLTV MYIMLKNLVNVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGIIKTEFDKSFAENAA RYLRNARWY KLILDNLKKSERAWNEFRNTVCHLNAIRNI INIKEI KEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYC KDFVKAYCTPFGYNLVRYKNLTIDGLFDKNYPGKDDSDEQK (SEQ ID NO: 1) >WP_046441786.1 (RspCasl3d)
[ Ruminococcus sp. N15.MGS-57]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVL EYEVDNNDYNQTQLSSKGSSNIELRGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDD NIHIQLIYNILDIEKILAVYVTNIVYALNNMLSIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQI RQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKG FIQGNKVNISLLI DMMKGYEADDII RLYYDFIVLKSQKNLGFSI KKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNY YRNDWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKI LDSEKKNASDLLYF SKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLF DSQRITNELFIVKNIASMRKPASSAKL TMFRDALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYAAQKIRKVAKNEKWMFVLGGIPDT QIERYYKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVI AIHCLERDFGLYKEIIPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWR ELKEYIGDIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRN EYLTEK ( SEQ ID NO: 2)
In some embodiments, the CRISPR-associated proteins described herein (e.g., Type VI-D CRISPR-Cas effector proteins) are from about 800 to about 1150 amino acids long, such as about 850 to about 1100 amino acids in length, e.g., about 850 to about 1050, about 850 to about 1000 amino acids long, or about 850 to about 950 amino acids long.
In some embodiments, the CRISPR-associated proteins (e.g., Type VI-D CRISPR- Cas effector proteins) have RNAse activity (e.g., collateral RNAse activity). In some embodiments, the CRISPR-associated proteins have DNAse activity. In some embodiments, the DNAse and/or RNAse activity is mediated by a single or both HEPN domains present in the CRISPR-associated proteins.
In some embodiments, a CRISPR-associated protein (e.g., Type VI-D CRISPR-Cas effector protein) is derived from a Ruminococcus or Eubacterium bacterium. In some embodiments, the CRISPR associated protein is derived from a human stool sample bacterial source.
Collateral RNase Activity
In some embodiments, a complex comprised of (but not limited to) a CRISPR- associated protein and a crRNA is activated upon binding to a target nucleic acid (e.g., a target RNA). Activation induces a conformational change, which results in the complex acting as a non-specific RNase, cleaving and/or degrading nearby RNA molecules (e.g., ssRNA or dsRNA molecules) (i.e. , "collateral" effects).
Collateral-free RNA Cleavage
In other embodiments, a complex comprised of (but not limited to) the CRISPR- associated protein and a crRNA does not exhibit collateral RNase activity subsequent to target recognition. This "collateral-free" embodiment may comprise wild-type or engineered effector proteins.
PAM/PFS-Independent Targeting
In some embodiments, a CRISPR-associated protein (e.g., a Type VI-D CRISPR-Cas effector protein described herein) recognizes and cleaves the target nucleic acid without any additional requirements adjacent to or flanking the protospacer (i.e., protospacer adjacent motif "PAM" or protospacer flanking sequence "PFS" requirements).
Deactivated/Inactivated CRISPR-Associated Proteins
Where the CRISPR-associated proteins described herein have nuclease activity, the CRISPR-associated proteins can be modified to have diminished nuclease activity, e.g., nuclease inactivation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type CRISPR-associated proteins. The nuclease activity can be diminished by several methods known in the art, e.g., introducing mutations into the nuclease domains of the proteins. In some embodiments, catalytic residues for the nuclease activities are identified, and these amino acid residues can be substituted by different amino acid residues (e.g., glycine or alanine) to diminish the nuclease activity. In some embodiments, the amino acid substitution is a conservative amino acid substitution. In some embodiments, the amino acid substitution is a non-conservative amino acid substitution.
In some embodiments, the CRISPR-associated proteins described herein (e.g., a Type VI-D CRISPR-Cas effector protein) are modified to comprise one or more mutations (e.g., amino acid deletions, insertions, or substitutions) in at least one HEPN domain. In some embodiments, the CRISPR associate protein includes one, two, three, four, five, six, seven, eight, nine, or more amino acid substitutions in at least one HEPN domain. For example, in some embodiments, the one or more mutations comprise asubstitution (e.g., an alanine substitution) at an amino acid residue corresponding to R295, H300, R849, H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2. The presence of at least one of these mutations results in a CRISPR-associated protein having reduced nuclease activity (e.g., RNAse activity) as compared to the nuclease activity of the CRISPR-associated protein from which the protein was derived (i.e., lacking the mutation).
The inactivated CRISPR-associated proteins can be fused or associated with one or more functional domains (e.g., via fusion protein, linker peptides, "GS" linkers, etc.). These functional domains can have various activities, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, base-editing activity, and switch activity (e.g., light inducible). In some embodiments, the functional domains are Kruppel associated box (KRAB), VP64, VP16, Fokl, P65, HSF1, MyoDl, Adenosine Deaminase Acting on RNA (ADAR) 1, ADAR2, APOBEC, cytidine deaminase (AID), mini-SOG, APEX, and biotin-APEX. In some embodiments, the functional domain is a base editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID). In some embodiments, the CRISPR-associated protein is fused to one functional domain. In some embodiments, the CRISPR-associated protein is fused to multiple (e.g., two, three, four, five, six, seven, eight, or more) functional domains. In some embodiments, the functional domain (e.g., a base editing domain) is further fused to an RNA- binding domain (e.g., MS2). In some embodiments, the CRISPR-associated protein is associated to or fused to a functional domain via a linker sequence (e.g., a flexible linker sequence or a rigid linker sequence). Exemplary linker sequences and functional domain sequences are provided in Table 10.
The positioning of the one or more functional domains on the inactivated CRISPR- associated proteins is one that allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. For example, if the functional domain is a transcription activator (e.g., VP16, VP64, or p65), the transcription activator is placed in a spatial orientation that allows it to affect the transcription of the target. Likewise, a transcription repressor is positioned to affect the transcription of the target, and a nuclease (e.g., Fokl) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is positioned at the N-terminus of the CRISPR-associated protein. In some embodiments, the functional domain is positioned at the C-terminus of the CRISPR- associated protein. In some embodiments, the inactivated CRISPR-associated protein is modified to comprise a first functional domain at the N-terminus and a second functional domain at the C-terminus.
Various examples of inactivated CRISPR-associated proteins fused with one or more functional domains and methods of using the same are described, e.g., in International Publication No. WO 2017/219027, which is incorporated herein by reference in its entirety, and in particular with respect to the features described herein. Split Enzymes
The present disclosure also provides a split version of the CRISPR-associated proteins described herein (e.g., a Type VI-D CRISPR-Cas effector protein). The split version of the CRISPR-associated protein may be advantageous for delivery. In some embodiments, the CRISPR-associated proteins are split into two parts of the enzyme, which together substantially comprise a functioning CRISPR-associated protein.
The split can be done in a way that the catalytic domain(s) are unaffected. The CRISPR-associated protein may function as a nuclease or may be an inactivated enzyme, which is essentially a RNA-binding protein with very little or no catalytic activity (e.g., due to mutation(s) in its catalytic domains). Split enzymes are described, e.g., in Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat 'l. Acad. Sci. , 112.10 (2015): 2984-2989, which is incorporated herein by reference in its entirety.
In some embodiments, the nuclease lobe and a-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the crRNA recruits them into a ternary complex that recapitulates the activity of full-length CRISPR-associated proteins and catalyzes site-specific DNA cleavage. The use of a modified crRNA abrogates split-enzyme activity by preventing dimerization, allowing for the development of an inducible dimerization system.
In some embodiments, the split CRISPR-associated protein can be fused to a dimerization partner, e.g., by employing rapamycin sensitive dimerization domains. This allows the generation of a chemically inducible CRISPR-associated protein for temporal control of the activity of the protein. The CRISPR-associated protein can thus be rendered chemically inducible by being split into two fragments and rapamycin-sensitive dimerization domains can be used for controlled re-assembly of the protein.
The split point is typically designed in silico and cloned into the constructs. During this process, mutations can be introduced to the split CRISPR-associated protein and nonfunctional domains can be removed. In some embodiments, the two parts or fragments of the split CRISPR-associated protein (i.e., the N-terminal and C-terminal fragments), can form a full CRISPR-associated protein, comprising, e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the sequence of the wild-type CRISPR-associated protein.
Self-Activating or Inactivating Enzymes
The CRISPR-associated proteins described herein (e.g., a Type VI-D CRISPR-Cas effector protein) can be designed to be self-activating or self-inactivating. For example, the target sequence can be introduced into the coding construct of the CRISPR-associated protein. Thus, the CRISPR-associated protein can cleave the target sequence, as well as the construct encoding the protein thereby self-inactivating their expression. Methods of constructing a self-inactivating CRISPR system are described, e.g., in Epstein, and Schaffer, Mol. Ther. 24 (2016): S50, which is incorporated herein by reference in its entirety.
In some other embodiments, an additional crRNA, expressed under the control of a weak promoter (e.g., 7SK promoter), can target the nucleic acid sequence encoding the CRISPR-associated protein to prevent and/or block its expression (e.g., by preventing the transcription and/or translation of the nucleic acid). The transfection of cells with vectors expressing the CRISPR-associated protein, the crRNAs, and crRNAs that target the nucleic acid encoding the CRISPR-associated protein can lead to efficient disruption of the nucleic acid encoding the CRISPR-associated protein and decrease the levels of CRISPR-associated protein, thereby limiting the genome editing activity.
In some embodiments, the genome editing activity of the CRISPR-associated protein can be modulated through endogenous RNA signatures (e.g., miRNA) in mammalian cells. A CRISPR-associated protein switch can be made by using a miRNA-complementary sequence in the 5'-UTR of mRNA encoding the CRISPR-associated protein. The switches selectively and efficiently respond to miRNA in the target cells. Thus, the switches can differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Therefore, the switch systems can provide a framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information (see, e.g., Hirosawa et al. Nucl. Acids Res., 2017, 45(13): el 18).
Inducible CRISPR-associated proteins
The CRISPR-associated proteins (e.g., Type VI-D CRISPR-Cas effector proteins) can be inducibly expressed, e.g., their expression can be light-induced or chemically-induced. This mechanism allows for activation of the functional domain in the CRISPR-associated proteins. Light inducibility can be achieved by various methods known in the art, e.g., by designing a fusion complex wherein CRY2PHR/CIBN pairing is used in split CRISPR- associated proteins (see, e.g., Konermann et al. "Optical control of mammalian endogenous transcription and epigenetic states," Nature, 500.7463 (2013): 472). Chemical inducibility can be achieved, e.g., by designing a fusion complex wherein FKBP/FRB (FK506 binding protein / FKBP rapamycin binding domain) pairing is used in split CRISPR-associated proteins. Rapamycin is required for forming the fusion complex, thereby activating the CRISPR-associated proteins (see, e.g., Zetsche, Volz, and Zhang, "A split-Cas9 architecture for inducible genome editing and transcription modulation," Nature Biotech., 33.2 (2015): 139-142).
Furthermore, expression of the CRISPR-associated proteins can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression system), hormone inducible gene expression system (e.g., an ecdysone inducible gene expression system), and an arabinose-inducible gene expression system. When delivered as RNA, expression of the RNA targeting effector protein can be modulated via a riboswitch, which can sense a small molecule like tetracycline (see, e.g., Goldfiess, Stephen J. et al. "Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction," Nucl. Acids Res., 40.9 (2012): e64-e64).
Various embodiments of inducible CRISPR-associated proteins and inducible CRISPR systems are described, e.g., in US Patent No. 8,871,445, US Publication No.
2016/0208243, and International Publication No. WO 2016/205764, each of which is incorporated herein by reference in its entirety.
Functional Mutations
In some embodiments, the CRISPR-associated proteins include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Localization Signal (NLS) attached to the N-terminal or C- terminal of the protein. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence
PKKKRKV (SEQ ID NO: 135); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 136)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 137) or
RQRRNELKRSP (SEQ ID NO: 138); the hRNPAl M9 NLS having the sequence
NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 139); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 140) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 141) and PPKKARED (SEQ ID NO: 142) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 143) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 144) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 145) and PKQKKRK(SEQ ID NO: Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 148) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 149) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 150) of the human glucocorticoid receptor. In some embodiments, the CRISPR- associated protein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Export Signal (NES) attached the N-terminal or C-terminal of the protein. In a preferred embodiment a C-terminal and/or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.
In some embodiments, the CRISPR-associated proteins described herein are mutated at one or more amino acid residues to alter one or more functional activities. For example, in some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its helicase activity. In some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its nuclease activity (e.g., endonuclease activity or exonuclease activity). In some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with an RNA guide. In some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid.
In some embodiments, the CRISPR-associated proteins described herein are capable of cleaving a target nucleic acid molecule. In some embodiments, the CRISPR-associated protein cleaves both strands of the target nucleic acid molecule. However, in some embodiments, the CRISPR-associated protein is mutated at one or more amino acid residues to alter its cleaving activity. For example, in some embodiments, the CRISPR-associated protein may comprise one or more mutations that render the enzyme incapable of cleaving a target nucleic acid. In other embodiments, the CRISPR-associated protein comprise one or more mutations such that the enzyme is capable of cleaving a single strand of the target nucleic acid (i.e. , nickase activity). In some embodiments, the CRISPR-associated protein is capable of cleaving the strand of the target nucleic acid that is complementary to the strand to which the RNA guide hybridizes. In some embodiments, the CRISPR-associated protein is capable of cleaving the strand of the target nucleic acid to which the guide RNA hybridizes.
In some embodiments, a CRISPR-associated protein described herein can be engineered to have a deletion in one or more amino acid residues to reduce the size of the enzyme while retaining one or more desired functional activities (e.g., nuclease activity and the ability to interact functionally with a RNA guide). The truncated CRISPR-associated protein can be advantageously used in combination with delivery systems having load limitations.
Nucleic acids encoding the proteins (e.g., a CRISPR-associated protein or an accessory protein) and RNA guides (e.g., a crRNA) described herein are also provided. In some embodiments, the nucleic acid is a synthetic nucleic acid. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule). In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methylcytidine, substituted with pseudouridine, or a combination thereof. In some embodiments, the nucleic acid (e.g., DNA) is operably linked to a regulatory element (e.g., a promoter) in order to control the expression of the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter. Suitable promoters are known in the art and include, for example, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a HI promoter, retroviral Rous sarcoma virus LTR promoter, a
cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, and a β-actin promoter. For example, a U6 promoter can be used to regulate the expression of an RNA guide molecule described herein.
In some embodiments, the nucleic acid(s) are present in a vector (e.g., a viral vector or a phage). The vectors can include one or more regulatory elements that allow for the propagation of the vector in a cell of interest (e.g., a bacterial cell or a mammalian cell). In some embodiments, the vector includes a nucleic acid encoding a single component of a CRISPR-associated (Cas) system described herein. In some embodiments, the vector includes multiple nucleic acids, each encoding a component of a CRISPR-associated (Cas) system described herein.
In one aspect, the present disclosure provides nucleic acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequences described herein. In another aspect, the present disclosure also provides amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences described herein.
In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100
nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as the sequences described herein. In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from the sequences described herein.
In some embodiments, the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as the sequences described herein. In some embodiments, the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from the sequences described herein.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In general, the length of a reference sequence aligned for comparison purposes should be at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
In some embodiments, the CRISPR-associated proteins and accessory proteins described herein can be fused to one or more peptide tags, including a His-tag, GST-tag, or myc-tag. In some embodiments, the CRISPR-associated proteins or accessory proteins described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein or yellow fluorescent protein).
The proteins described herein (e.g., CRISPR-associated proteins or accessory proteins) can be delivered or used as either nucleic acid molecules or polypeptides. When nucleic acid molecules are used, the nucleic acid molecule encoding the CRISPR-associated proteins can be codon-optimized. The nucleic acid can be codon optimized for use in any organism of interest, in particular human cells or bacteria. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA).
RNA Guides
In some embodiments, the CRISPR systems described herein include at least RNA guide (e.g., a crRNA). The architecture of multiple RNA guides is known in the art (see, e.g., International Publication Nos. WO 2014/093622 and WO 2015/070083, the entire contents of each of which are incorporated herein by reference). In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., one, two, three, four, five, six, seven, eight, or more RNA guides). In some embodiments, the RNA guide includes a crRNA. In some embodiments, the RNA guide includes a crRNA and a tracrRNA. In some embodiments, the RNA guide is an engineered construct that includes a tracrRNA and a crRNA (in a single RNA guide). Sequences for RNA guides from multiple CRISPR systems are known in the art and can be searched using public databases (see, e.g., Grissa et al. (2007) Nucleic Acids Res. 35 (web server issue): W52-7; Grissa et al. (2007) BMC Bioinformatics 8: 172; Grissa et al. (2008) Nucleic Acids Res. 36 (web server issue): W145-8; and Moller and Liang (2017) PeerJ 5: e3788; see also the CRISPR database available at:
crispr.i2bc.paris-saclay.fr/crispr/BLAST/CRISPRsBlast.php; and MetaCRAST available at: github.com/molleraj/MetaCRAST).
In some embodiments, the CRISPR systems described herein include at least one crRNA or a nucleic acid encoding at least one crRNA. In some embodiments, the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence, which is typical of precursor crRNA (pre-crRNA) configurations in other CRISPR systems. In some embodiments, the crRNA includes a truncated direct repeat sequence and a spacer sequence, which is typical of processed or mature crRNA. The CRISPR-associated protein is capable of cleaving pre-crRNA to form processed or mature crRNA. The CRISPR-associated protein forms a complex with the mature crRNA, and the spacer sequence directs the complex to a sequence-specific binding with the target nucleic acid that is complementary to the spacer sequence. The resulting complex comprises the CRISPR-associated protein and the mature crRNA bound to the target RNA.
In some embodiments, the CRISPR systems described herein include a mature crRNA. In some embodiments, the CRISPR systems described herein include a pre-crRNA.
In some embodiments, the CRISPR systems described herein include a plurality of crRNAs (e.g., 2, 3, 4, 5, 10, 15, or more) or a plurality of nucleic acids encoding a plurality of crRNAs. Generally, the crRNAs described herein include a direct repeat sequence and a spacer sequence. In certain embodiments, the crRNA includes, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence.
In some embodiments, the CRISPR system described herein includes an RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 151) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is G or T, X3 is A or G, X4 is C or G or T, X5 is C or T, and Χβ is A or G. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'- X1X2X3X4TX5TX6 AAAC-3 ' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is C or T, and Χβ is A or G .
Exemplary RNA guide direct repeat sequences and effector protein pairs are provided in Table 3. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence listed in Table 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589). In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial three 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial four 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial five 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial six 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial seven 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 3 with a truncation of the initial eight 5' nucleotides.
In some embodiments, the direct repeat sequence comprises or consists of the nucleic acid sequence 5'-GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 34) or 5'-CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 72). In some embodiments, the direct repeat sequence comprises or consists of the nucleic acid sequence 5'-CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'-CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
In some embodiments, the CRISPR-associated protein comprises the amino acid sequence of SEQ ID NO: 1 and the crRNA comprises a direct repeat sequence, wherein the direct repeat sequence comprises or consists of the nucleic acid sequence 5'- GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 34) or 5'- CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152). In some embodiments, the CRISPR-associated protein comprises the amino acid sequence of SEQ ID NO: 2 and the crRNA comprises a direct repeat sequence, wherein the direct repeat sequence comprises or consists of the nucleic acid sequence 5'- CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 72) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
Multiplexing RNA Guides
Type VI CRISPR-Cas effectors have been demonstrated to employ more than one RNA guide, thus enabling the ability of these effectors, and systems and complexes that include them, to target multiple nucleic acids. In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more) RNA guides. In some embodiments, the CRISPR systems described herein include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem. The single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof. The processing capability of the Type VI-D CRISPR-Cas effector proteins described herein enables these effectors to be able to target multiple target nucleic acids (e.g., target RNAs) without a loss of activity. In some embodiments, the Type VI-D CRISPR-Cas effector proteins may be delivered in complex with multiple RNA guides directed to different target nucleic acids. In some embodiments, the Type VI-D CRISPR-Cas effector proteins may be co-delivered with multiple RNA guides, each specific for a different target nucleic acid. Methods of multiplexing using CRISPR-associated proteins are described, for example, in US 9,790, 490 B2, and EP 3009511 Bl, the entire contents of each of which are expressly incorporated herein by reference.
Spacer Lengths
The spacer length of crRNAs can range from about 15 to 50 nucleotides. In some embodiments, the spacer length of an RNA guide is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides. In some embodiments, the spacer length is from 15 to 17 nucleotides (e.g., 15, 16, or 17 nucleotides), from 17 to 20 nucleotides (e.g., 17, 18, 19, or 20 nucleotides), from 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), from 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), from 24 to 27 nucleotides, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides), from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides), or longer. In some embodiments, the direct repeat length of the RNA guide is at least 16 nucleotides, or is from 16 to 20 nucleotides (e.g., 16, 17, 18, 19, or 20 nucleotides). In some embodiments, the spacer length is from about 15 to about 42 nucleotides. In some embodiments, the direct repeat length of the RNA guide is 19 nucleotides.
The crRNA sequences can be modified in a manner that allows for formation of a complex between the crRNA and CRISPR-associated protein and successful binding to the target, while at the same time not allowing for successful nuclease activity (i.e., without nuclease activity / without causing indels). These modified guide sequences are referred to as "dead crRNAs," "dead guides," or "dead guide sequences." These dead guides or dead guide sequences may be catalytically inactive or conformationally inactive with regard to nuclease activity. Dead guide sequences are typically shorter than respective guide sequences that result in active RNA cleavage. In some embodiments, dead guides are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective RNA guides that have nuclease activity. Dead guide sequences of RNA guides can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length), from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length).
Thus, in one aspect, the disclosure provides non-naturally occurring or engineered CRISPR systems including a functional CRISPR-associated protein as described herein, and a crRNA, wherein the crRNA comprises a dead crRNA sequence whereby the crRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a genomic locus of interest in a cell without detectable nuclease activity (e.g., RNAse activity).
A detailed description of dead guides is described, e.g., in International Publication No. WO 2016/094872, which is incorporated herein by reference in its entirety.
Inducible Guides
RNA guides (e.g., crRNAs) can be generated as components of inducible systems. The inducible nature of the systems allows for spatio-temporal control of gene editing or gene expression. In some embodiments, the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.
In some embodiments, the transcription of RNA guides (e.g., crRNA) can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems), and arabinose-inducible gene expression systems. Other examples of inducible systems include, e.g., small molecule two- hybrid transcription activations systems (FKBP, ABA, etc.), light inducible systems
(Phytochrome, LOV domains, or cryptochrome), or Light Inducible Transcriptional Effector (LITE). These inducible systems are described, e.g., in WO 2016205764 and US 8795965, both of which are incorporated herein by reference in the entirety.
Chemical Modifications
Chemical modifications can be applied to the crRNA's phosphate backbone, sugar, and/or base. Backbone modifications such as phosphorothioates modify the charge on the phosphate backbone and aid in the delivery and nuclease resistance of the oligonucleotide (see, e.g., Eckstein, "Phosphorothioates, essential components of therapeutic
oligonucleotides," Nucl. Acid Ther., 24 (2014), pp. 374-387); modifications of sugars, such as 2'-0-methyl (2'-OMe), 2'-F, and locked nucleic acid (LNA), enhance both base pairing and nuclease resistance (see, e.g., Allerson et al. "Fully 2 '-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA," J. Med. Chem., 48.4 (2005): 901-904). Chemically modified bases such as 2- thiouridine or N6-methyladenosine, among others, can allow for either stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering," Front. Genet., 2012 Aug 20; 3: 154). Additionally, RNA is amenable to both 5' and 3' end conjugations with a variety of functional moieties including fluorescent dyes, polyethylene glycol, or proteins.
A wide variety of modifications can be applied to chemically synthesized crRNA molecules. For example, modifying an oligonucleotide with a 2'-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing. Furthermore, a 2'- OMe modification can affect how the oligonucleotide interacts with transfection reagents, proteins or any other molecules in the cell. The effects of these modifications can be determined by empirical testing.
In some embodiments, the crRNA includes one or more phosphorothioate modifications. In some embodiments, the crRNA includes one or more locked nucleic acids for the purpose of enhancing base pairing and/or increasing nuclease resistance.
A summary of these chemical modifications can be found, e.g., in Kelley et al, "Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing," J. Biotechnol. 2016 Sep 10; 233:74-83; WO 2016205764; and US 8795965 B2; each which is incorporated by reference in its entirety.
Sequence Modifications
The sequences and the lengths of the RNA guides (e.g., crRNAs) described herein can be optimized. In some embodiments, the optimized length of an RNA guide can be determined by identifying the processed form of crRNA (i.e., a mature crRNA), or by empirical length studies for crRNA tetraloops.
The crRNAs can also include one or more aptamer sequences. Aptamers are oligonucleotide or peptide molecules have a specific three-dimensional structure and can bind to a specific target molecule. The aptamers can be specific to gene effectors, gene activators, or gene repressors. In some embodiments, the aptamers can be specific to a protein, which in turn is specific to and recruits and/or binds to specific gene effectors, gene activators, or gene repressors. The effectors, activators, or repressors can be present in the form of fusion proteins. In some embodiments, the RNA guide has two or more aptamer sequences that are specific to the same adaptor proteins. In some embodiments, the two or more aptamer sequences are specific to different adaptor proteins. The adaptor proteins can include, e.g., MS2, PP7, QP, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, Mi l, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, <|>Cb5, <|>Cb8r, <|>Cbl2r, <|>Cb23r, 7s, and PRR1.
Accordingly, in some embodiments, the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein. In some
embodiments, the aptamer sequence is a MS2 binding loop (5'- ggcccAACAUGAGGAUCACCCAUGUCUGCAGgggcc-3' (SEQ ID NO: 169)). In some embodiments, the apatamer sequence is a QBeta binding loop (5'- ggcccAUGCUGUCUAAGACAGCAUgggcc-3' (SEQ ID NO: 170)). In some embodiments, the aptamer sequence is a PP7 binding loop (5'- ggcccUAAGGGUUUAUAUGGAAACCCUUAgggcc-3' (SEQ ID NO: 173)). A detailed description of aptamers can be found, e.g., in Nowak et al, "Guide RNA engineering for versatile Cas9 functionality," Nucl. Acid. Res., 2016 Nov 16;44(20):9555-9564; and WO 2016205764, which are incorporated herein by reference in their entirety. Target Nucleic Acids
The target nucleic acids can be a DNA molecule or a RNA molecule. As described above, in some embodiments, the CRISPR-associated proteins described herein have RNAse activity. Thus, the target nucleic acids can be any RNA molecule of interest, including naturally-occurring and engineered RNA molecules. The target RNA can be an mRNA, a tRNA, a ribosomal RNA (rRNA), a microRNA (miRNA), an interfering RNA (siRNA), a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA.
In some embodiments, the target nucleic acid is associated with a condition or disease (e.g., an infectious disease or a cancer). Thus, in some embodiments, the systems described herein can be used to treat a condition or disease by targeting these nucleic acids. For instance, the target nucleic acid associated with a condition or disease may be an RNA molecule that is overexpressed in a diseased cell (e.g., a cancer or tumor cell). The target nucleic acid may also be a toxic RNA and/or a mutated RNA (e.g., an mRNA molecule having a splicing defect or a mutation). The target nucleic acid may also be an RNA that is specific for a particular microorganism (e.g., a pathogenic bacteria).
Guide: Target Sequence Matching Requirements
In classic CRISPR systems, the degree of complementarity between a guide sequence (e.g., a crRNA) and its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of
complementarity is 100%. The RNA guides can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
To reduce off-target interactions, e.g., to reduce the guide interacting with a target sequence having low complementarity, mutations can be introduced to the CRISPR systems so that the CRISPR systems can distinguish between target and off-target sequences that have greater than 80%, 85%, 90%, or 95% complementarity. In some embodiments, the degree of complementarity is from 80% to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches). Accordingly, in some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity is 100%. It is known in the field that complete complementarity is not required, provided there is sufficient complementarity to be functional. Modulations of cleavage efficiency can be exploited by introduction of mismatches, e.g., one or more mismatches, such as 1 or 2 mismatches between spacer sequence and target sequence, including the position of the mismatch along the spacer/target. The more central (i.e., not at the 3' or 5' ends) a mismatch, e.g., a double mismatch, is located; the more cleavage efficiency is affected. Accordingly, by choosing mismatch positions along the spacer sequence, cleavage efficiency can be modulated. For example, if less than 100% cleavage of targets is desired (e.g., in a cell population), 1 or 2 mismatches between spacer and target sequence can be introduced in the spacer sequences.
Target Ncleic Acids to Regulate Collateral RNAse Activity Activation
In some embodiments, the CRISPR systems described herein further comprise a target nucleic acid (e.g., a linear or circular nucleic acid) which may advantageously be used to activate the collateral RNAse activity of a Type VI-D CRISPR-Cas effector protein in a controlled manner. By regulating the expression and/or delivery of the target nucleic acid, the activation of the collateral RNAse activity of the effector protein may be controlled. For example, exogenous target nucleic acid may be included in the system to increase the activation rate of the collateral RNAse activity of a Type VI-D CRISPR-Cas effector protein. In some embodiments, the target nucleic acid is a DNA molecule. In some embodiments, the target nucleic acid is an RNA molecule (e.g., a mRNA molecule). In some embodiments, when the target nucleic acid is an RNA, the system includes a DNA molecule (e.g., a plasmid DNA) that codes for the target nucleic acid that is specifically targeted by the Type VI-D CRISPR-Cas effector protein and crRNA complex, operably linked to a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter.
Accessory Proteins
In one aspect, the CRISPR systems described herein includes at least one accessory protein. As shown in Example 4, the inventors have surprisingly discovered that the accessory proteins described herein enhance the nuclease activity of CRISPR-associated proteins (e.g., Type VI-D CRISPR-Cas effector proteins) as compared to the nuclease activity of the CRISPR associated protein in the absence of the accessory protein. The ability of the accessory proteins described herein to enhance the nuclease activity of CRISPR-associated proteins is particularly desireable in clinical and therapeutic applications. Therefore, CRISPR systems including at least one accessory protein are provided herein. For example, an accessory protein described herein may be used in combination with CRISPR-associated proteins known in the art in order to enhance their nuclease activity. Alternatively, an accessory protein may be used in combination with a Type VI-D CRISPR-Cas effector protein described herein to enhance its nuclease activity (e.g., collateral RNAse activity or targeted RNAse activity).
In some embodiments, the accessory protein includes a WYL domain (PFAM:
PF 13280), which has been predicted to be a ligand-sensing domain, which can regulate CRISPR-Cas systems. WYL domains are SH3 beta-barrel fold containing domains named for three conserved amino acids found in some domains belonging to the WYL-like superfamily. One WYL domain protein, sll7009, has been found to be a negative regulator of the Synechocystis sp. I-D CRISPR-Cas system (see, e.g., Hein et al. (2013) RNA Biol. 10: 852-64).
In some embodiments, the accessory protein includes at least one WYL domain. In some embodiments, the accessory protein includes two WYL domains. In some
embodiments, the accessory protein includes a helix-turn-helix (HTH) fold. In some embodiments, the accessory protein includes a ribbon-helix-helix (RHH) fold. In some embodiments, the accessory protein includes at least one WYL domain, wherein the WYL domain comprises the amino acid sequence PXXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid. In some embodiments, the accessory protein includes at least one WYL domain, wherein the WYL domain comprises the amino acid sequence PXXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid; and at least one ribbon-ribbon-helix (RHH) fold or at least one helix-turn-helix (HTH) domain. In some embodiments, the amino acid sequence of the WYL domain is separate from (i.e., does not overlap with) an RHH fold or an HTH fold.
In some embodiments, the accessory proteins describe herein modulate the RNAse activity of a CRISPR-associated protein. In some embodiments, the accessory protein modulates (e.g., increases or decreases) the collateral RNAse activity of a CRISPR-associated protein. In some embodiments, the accessory protein modulates (e.g., increases or decreases) the RNA-binding activity of a CRISPR-associated protein. In some embodiments, the accessory protein modulates (e.g., increases or decreases) the crRNA processing activity of a CRISPR-associated protein. In some embodiments, the accessory protein modulates (e.g., increases or decreases) the targeted RNAse activity of a CRISPR-associated protein.
In some embodiments, the accessory proteins described herein enhances the RNAse activity of a CRISPR-associated protein (e.g., a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, a Cas9 protein). In some embodiments, the accessory protein enhances the collateral RNAse activity of a CRISPR-associated protein. In some embodiments, the accessory protein enhances the crRNA processing activity of a CRISPR- associated protein. In some embodiments, the accessory protein enhances the RNA-binding activity of a CRISPR-associated protein. In some embodiments, the accessory protein enhances the targeted RNAse activity of a CRISPR-associated protein. CRISPR systems comprising an accessory protein described herein are particularly useful in applications where increased sequence-specific or collateral RNA degradation is desireable. For example, in diagnostic applications, enhanced RNAse activity provides a greater degree of sensitivity, allowing the detection of lower concentrations of a target RNA. In some embodiments, an accessory protein described herein enhances the RNAse activity of the ternary complex of multiple CRISPR Type VI effectors. The ability of the accessory protein to enhance the RNAse of multiple effectors is particularly useful in applications where combinations of Type VI effectors of different sub-types are used together, for example in multi-channel diagnostic applications. In some embodiments, the accessory protein can enhance the RNAse activity of Type VI effectors outside the Casl3d family thereby providing a valuable tool for screening the activity of uncharacterized Type VI effectors.
Exemplary accessory proteins are provided below in Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory proteins include an amino acid sequence having at least about 80% identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequence of any one of Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein includes the amino acid sequence of any one of the proteins in Tables 4, 5 and 6 (e.g., SEQ ID NOs. 78-93, and 590-671). In some embodiments, the accessory protein is RspWYLl (SEQ ID NO: 81). Methods of Using CRISPR Systems
The CRISPR systems described herein have a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, or activating) a target polynucleotide or nucleic acid in a multiplicity of cell types. The CRISPR systems have a broad spectrum of applications in, e.g., DNA/RNA detection (e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK)), tracking and labeling of nucleic acids, enrichment assays (extracting desired sequence from background), controlling interfering RNA or miRNA, detecting circulating tumor DNA, preparing next generation library, drug screening, disease diagnosis and prognosis, and treating various genetic disorders.
DNA RNA Detection
In one aspect, the CRISPR systems described herein can be used in DNA or RNA detection. CRISPR-associated proteins can be reprogrammed with CRISPR RNAs (crRNAs) to provide a platform for specific RNA sensing. Upon recognition of its RNA target, activated CRISPR-associated proteins engage in "collateral" cleavage of nearby non-targeted RNAs. This crRNA-programmed collateral cleavage activity allows the CRISPR systems to detect the presence of a specific RNA by triggering programmed cell death or by nonspecific degradation of labeled RNA.
The SHERLOCK method (Specific High Sensitivity Enzymatic Reporter
UnLOCKing) provides an in vitro nucleic acid detection platform with attomolar sensitivity based on nucleic acid amplification and collateral cleavage of a reporter RNA, allowing for real-time detection of the target. To achieve signal detection, the detection can be combined with different isothermal amplification steps. For example, recombinase polymerase amplification (RPA) can be coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection. The combination of amplification by RPA, T7 RNA polymerase transcription of amplified DNA to RNA, and detection of target RNA by collateral RNA cleavage-mediated release of reporter signal is referred as SHERLOCK. Methods of using CRISPR in SHERLOCK are described in detail, e.g., in Gootenberg, et al. "Nucleic acid detection with CRISPR-Casl3a/C2c2," Science, 2017 Apr 28;356(6336):438-442, which is incorporated herein by reference in its entirety.
The CRISPR-associated proteins can further be used in Northern blot assays, which use electrophoresis to separate RNA samples by size. The CRISPR-associated proteins can be used to specifically bind and detect the target RNA sequence. The CRISPR-associated proteins can also be fused to a fluorescent protein (e.g., GFP) and used to track RNA localization in living cells. More particularly, the CRISPR-associated proteins can be inactivated in that they no longer cleave RNAs as described above. Thus, CRISPR-associated proteins can be used to determine the localization of the RNA or specific splice variants, the level of mRNA transcripts, up- or down- regulation of transcripts and disease-specific diagnosis. The CRISPR-associated proteins can be used for visualization of RNA in (living) cells using, for example, fluorescent microscopy or flow cytometry, such as fluorescence- activated cell sorting (FACS), which allows for high-throughput screening of cells and recovery of living cells following cell sorting. A detailed description regarding how to detect DNA and RNA can be found, e.g., in International Publication No. WO 2017/070605, which is incorporated herein by reference in its entirety.
In some embodiments, the CRISPR systems described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH). These methods are described in, e.g., Chen et al, "Spatially resolved, highly multiplexed RNA profiling in single cells," Science, 2015 Apr 24; 348(6233):aaa6090, which is incorporated herein by reference herein in its entirety.
In some embodiments, the CRISPR systems described herein can be used to detect a target RNA in a sample (e.g., a clinical sample, a cell, or a cell lysate). The collateral RNAse activity of the Type VI-D CRISPR-Cas effector proteins described herein is activated when the effector proteins bind to a target nucleic acid. Upon binding to the target RNA of interest, the effector protein cleaves a labeled detector RNA to generate a signal (e.g., an increased signal or a decreased signal) thereby allowing for the qualitative and quantitative detection of the target RNA in the sample. The specific detection and quantification of RNA in the sample allows for a multitude of applications including diagnostics. In some embodiments, the methods include contacting a sample with: i) an RNA guide (e.g., crRNA) and/or a nucleic acid encoding the RNA guide, wherein the RNA guide consists of a direct repeat sequence and a spacer sequence capable of hybridizing to the target RNA; (ii) a Type VI-D CRISPR-Cas effector protein and/or a nucleic acid encoding the effector protein; and (iii) a labeled detector RNA; wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the single- stranded target RNA in the sample. In some embodiments, the methods further comprise comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample. In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing. In some embodiments, the labeled detector RNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair. In some embodiments, upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is decreased or increased. In some embodiments, the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein. In some embodiments, a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein. In some embodiments, the labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof. In some embodiments, the methods include the multi-channel detection of multiple independent target RNAs in a sample (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more target RNAs) by using multiple Type VI-D CRISPR-Cas systems, each including a distinct orthologous effector protein and corresponding RNA guides, allowing for the differentiation of multiple target RNAs in the sample. In some embodiments, the methods include the multi-channel detection of multiple independent target RNAs in a sample, with the use of multiple instances of Type VI-D CRISPR-Cas systems, each containing an orthologous effector protein with differentiable collateral RNAse substrates. Methods of detecting an RNA in a sample using CRISPR-associated proteins are described, for example, in U.S. Patent Publication No. 2017/0362644, the entire contents of which are incorporated herein by reference.
Tracking and Labeling of Nucleic Acids
Cellular processes depend on a network of molecular interactions among proteins, RNAs, and DNAs. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques employ an affinity tag combined with, a reporter group, e.g., a photoactivatable group, to label polypeptides and RNAs in the vicinity of a protein or RNA of interest in vitro. After UV irradiation, the photoactivatable groups react with proteins and other molecules that are in close proximity to the tagged molecules, thereby labelling them. Labelled interacting molecules can
subsequently be recovered and identified. The CRISPR-associated proteins can for instance be used to target probes to selected RNA sequences. These applications can also be applied in animal models for in vivo imaging of diseases or difficult-to culture cell types. The methods of tracking and labeling of nucleic acids are described, e.g., in US 8795965, WO
2016205764, and WO 2017070605; each of which is incorporated herein by reference herein in its entirety.
RNA Isolation, Purification, Enrichment, and/or Depletion
The CRISPR systems (e.g., CRISPR-associated proteins) described herein can be used to isolate and/or purify the RNA. The CRISPR-associated proteins can be fused to an affinity tag that can be used to isolate and/or purify the RNA-CRISPR-associated protein complex. These applications are useful, e.g., for the analysis of gene expression profiles in cells.
In some embodiments, the CRISPR-associated proteins can be used to target a specific noncoding RNA (ncRNA) thereby blocking its activity. In some embodiments, the CRISPR-associated proteins can be used to specifically enrich a particular RNA (including but not limited to increasing stability, etc.), or alternatively, to specifically deplete a particular RNA (e.g., particular splice variants, isoforms, etc.).
These methods are described, e.g., in US 8795965, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference herein in its entirety.
High-Throughput Screening
The CRISPR systems described herein can be used for preparing next generation sequencing (NGS) libraries. For example, to create a cost-effective NGS library, the CRISPR systems can be used to disrupt the coding sequence of a target gene, and the CRISPR- associated protein transfected clones can be screened simultaneously by next-generation sequencing (e.g., on the Ion Torrent PGM system). A detailed description regarding how to prepare NGS libraries can be found, e.g., in Bell et al, "A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing," BMC Genomics, 15.1 (2014): 1002, which is incorporated herein by reference in its entirety. Engineered Microorganisms
Microorganisms (e.g., E. coli, yeast, and microalgae) are widely used for synthetic biology. The development of synthetic biology has a wide utility, including various clinical applications. For example, the programmable CRISPR systems can be used to split proteins of toxic domains for targeted cell death, e.g., using cancer-linked RNA as target transcript. Further, pathways involving protein-protein interactions can be influenced in synthetic biological systems with e.g. fusion complexes with the appropriate effectors such as kinases or enzymes.
In some embodiments, crRNAs that target phage sequences can be introduced into the microorganism. Thus, the disclosure also provides methods of vaccinating a microorganism (e.g., a production strain) against phage infection.
In some embodiments, the CRISPR systems provided herein can be used to engineer microorganisms, e.g., to improve yield or improve fermentation efficiency. For example, the CRISPR systems described herein can be used to engineer microorganisms, such as yeast, to generate biofuel or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars. More particularly, the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and/or to modify endogenous genes, which may interfere with the biofuel synthesis. These methods of engineering microorganisms are described e.g., in Verwaal et al, "CRISPR/Cpfl enables fast and simple genome editing of Saccharomyces cerevisiae," Yeast, 2017 Sep 8. doi: 10.1002/yea.3278; and Hlavova et al., "Improving microalgae for biotechnology— from genetics to synthetic biology," Biotechnol. Adv., 2015 Nov 1; 33: 1194-203, both of which are incorporated herein by reference in the entirety.
In some embodiments, the CRISPR systems provided herein can be used to induce death or dormancy of a cell (e.g., a microorganism such as an engineered microorganism). These methods can be used to induce dormancy or death of a multitude of cell types including prokaryotic and eukaryotic cells, including, but not limited to mammalian cells (e.g., cancer cells, or tissue culture cells), protozoans, fungal cells, cells infected with a virus, cells infected with an intracellular bacteria, cells infected with an intracellular protozoan, cells infected with a prion, bacteria (e.g., pathogenic and non-pathogenic bacteria), protozoans, and unicellular and multicellular parasites. For instance, in the field of synthetic biology it is highly desireable to have mechanisms of controlling engineered microorganisms (e.g., bacteria) in order to prevent their propagation or dissemination. The systems described herein can be used as "kill-switches" to regulate and/or prevent the propagation or dissemination of an engineered microorganism. Further, there is a need in the art for alternatives to current antibiotic treatments. The systems described herein can also be used in applications where it is desirable to kill or control a specific microbial population (e.g., a bacterial population). For example, the systems described herein may include an RNA guide (e.g., a crRNA) that targets a nucleic acid (e.g., an RNA) that is genus-, species-, or strain- specific, and can be delivered to the cell. Upon complexing and binding to the target nucleic acid, the collateral RNAse activity of the Type VI-D CRISPR-Cas effector proteins is activated leading to the cleavage of non-target RNA within the microorganisms, ultimately resulting in dormancy or death.
In some embodiments, the methods comprise contacting the cell with a system described herein including a Type VI-D CRISPR-Cas effector proteins or a nucleic acid encoding the effector protein, and a RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the spacer sequence is complementary to at least 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides ) of a target nucleic acid (e.g., a genus-, strain-, or species-specific RNA guide). Without wishing to be bound by any particular theory, the cleavage of non-target RNA by the Type VI-D CRISPR-Cas effector proteins may induce programmed cell death, cell toxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, a reduction of cell growth, or a reduction in cell proliferation. For example, in bacteria, the cleavage of non- target RNA by the Type VI-D CRISPR-Cas effector proteins may be bacteriostatic or bacteriocidal.
Applications in Plants
The CRISPR systems described herein have a wide variety of utility in plants. In some embodiments, the CRISPR systems can be used to engineer genomes of plants (e.g., improving production, making products with desired post-translational modifications, or introducing genes for producing industrial products). In some embodiments, the CRISPR systems can be used to introduce a desired trait to a plant (e.g., with or without heritable modifications to the genome), or regulate expression of endogenous genes in plant cells or whole plants. In some embodiments, the CRISPR systems can be used to identify, edit, and/or silence genes encoding specific proteins, e.g., allergenic proteins (e.g., allergenic proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans). A detailed description regarding how to identify, edit, and/or silence genes encoding proteins is described, e.g., in Nicolaou et al, "Molecular diagnosis of peanut and legume allergy," Curr. Opin. Allergy Clin. Immunol, 2011 Jun; l l(3):222-8, and WO 2016205764 Al; both of which are incorporated herein by reference in the entirety.
Gene Drives
Gene drive is the phenomenon in which the inheritance of a particular gene or set of genes is favorably biased. The CRISPR systems described herein can be used to build gene drives. For example, the CRISPR systems can be designed to target and disrupt a particular allele of a gene, causing the cell to copy the second allele to fix the sequence. Because of the copying, the first allele will be converted to the second allele, increasing the chance of the second allele being transmitted to the offspring. A detailed method regarding how to use the CRISPR systems described herein to build gene drives is described, e.g., in Hammond et al, "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae " Nat. Biotechnol., 2016 Jan; 34(l):78-83, which is incorporated herein by reference in its entirety.
Pooled-Screening
As described herein, pooled CRISPR screening is a powerful tool for identifying genes involved in biological mechanisms such as cell proliferation, drug resistance, and viral infection. Cells are transduced in bulk with a library of RNA guide-encoding vectors described herein, and the distribution of RNA guides is measured before and after applying a selective challenge. Pooled CRISPR screens work well for mechanisms that affect cell survival and proliferation, and they can be extended to measure the activity of individual genes (e.g., by using engineered reporter cell lines). Arrayed CRISPR screens, in which only one gene is targeted at a time, make it possible to use RNA-seq as the readout. In some embodiments, the CRISPR systems as described herein can be used in single-cell CRISPR screens. A detailed description regarding pooled CRISPR screenings can be found, e.g., in Datlinger et al., "Pooled CRISPR screening with single-cell transcriptome read-out," Nat. Methods., 2017 Mar; 14(3):297-301, which is incorporated herein by reference in its entirety. Saturation Mutagenesis (Bashing)
The CRISPR systems described herein can be used for in situ saturating mutagenesis. In some embodiments, a pooled RNA guide library can be used to perform in situ saturating mutagenesis for particular genes or regulatory elements. Such methods can reveal critical minimal features and discrete vulnerabilities of these genes or regulatory elements (e.g., enhancers). These methods are described, e.g., in Canver et al, "BCL11 A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, 2015 Nov 12;
527(7577): 192-7, which is incorporated herein by reference in its entirety.
RNA-Related Applications
The CRISPR systems described herein can have various RNA-related applications, e.g., modulating gene expression, degrading a RNA molecule, inhibiting RNA expression, screening RNA or RNA products, determining functions of lincRNA or non-coding RNA, inducing cell dormancy, inducing cell cycle arrest, reducing cell growth and/or cell proliferation, inducing cell anergy, inducing cell apoptosis, inducing cell necrosis, inducing cell death, and/or inducing programmed cell death. A detailed description of these applications can be found, e.g., in WO 2016/205764 Al, which is incorporated herein by reference in its entirety. In different embodiments, the methods described herein can be performed in vitro, in vivo, or ex vivo.
For example, the CRISPR systems described herein can be administered to a subject having a disease or disorder to target and induce cell death in a cell in a diseased state (e.g., cancer cells or cells infected with an infectious agent). For instance, in some embodiments, the CRISPR systems described herein can be used to target and induce cell death in a cancer cell, wherein the cancer cell is from a subject having a Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non- Hodgkin's lymphoma, or urinary bladder cancer. Modulating Gene Expression
The CRISPR systems described herein can be used to modulate gene expression. The CRISPR systems can be used, together with suitable RNA guides, to target gene expression, via control of RNA processing. The control of RNA processing can include, e.g., RNA processing reactions such as RNA splicing (e.g., alternative splicing), viral replication, and tRNA biosynthesis. The RNA targeting proteins in combination with suitable RNA guides can also be used to control RNA activation (RNAa). RNA activation is a small RNA-guided and Argonaute (Ago)-dependent gene regulation phenomenon in which promoter-targeted short double-stranded RNAs (dsRNAs) induce target gene expression at the
transcriptional/epigenetic level. RNAa leads to the promotion of gene expression, so control of gene expression may be achieved that way through disruption or reduction of RNAa. In some embodiments, the methods include the use of the RNA targeting CRISPR as substitutes for e.g., interfering ribonucleic acids (such as siRNAs, shRNAs, or dsRNAs). The methods of modulating gene expression are described, e.g., in WO 2016205764, which is incorporated herein by reference in its entirety.
Controlling RNA Interference
Control over interfering RNAs or microRNAs (miRNA) can help reduce off-target effects by reducing the longevity of the interfering RNAs or miRNAs in vivo or in vitro. In some embodiments, the target RNAs can include interfering RNAs, i.e., RNAs involved in the RNA interference pathway, such as small hairpin RNAs (shRNAs), small interfering (siRNAs), etc. In some embodiments, the target RNAs include, e.g., miRNAs or double stranded RNAs (dsRNA).
In some embodiments, if the RNA targeting protein and suitable RNA guides are selectively expressed (for example spatially or temporally under the control of a regulated promoter, for example a tissue- or cell cycle-specific promoter and/or enhancer), this can be used to protect the cells or systems (in vivo or in vitro) from RNA interference (RNAi) in those cells. This may be useful in neighboring tissues or cells where RNAi is not required or for the purposes of comparison of the cells or tissues where the CRISPR-associated proteins and suitable crRNAs are and are not expressed (i.e., where the RNAi is not controlled and where it is, respectively). The RNA targeting proteins can be used to control or bind to molecules comprising or consisting of RNAs, such as ribozymes, ribosomes, or riboswitches. In some embodiments, the RNA guides can recruit the RNA targeting proteins to these molecules so that the RNA targeting proteins are able to bind to them. These methods are described, e.g., in WO 2016205764 and WO 2017070605, both of which are incorporated herein by reference in the entirety.
Modifying Riboswitches and Controlling Metabolic Regulations
Riboswitches are regulatory segments of messenger RNAs that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A specific riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Thus, in some embodiments, the riboswitch activity can be controlled by the use of the RNA targeting proteins in combination with suitable RNA guides to target the riboswitches. This may be achieved through cleavage of, or binding to, the riboswitch. Methods of using CRISPR systems to control riboswitches are described, e.g., in WO 2016205764 and WO
2017070605, both of which are incorporated herein by reference in their entireties.
RNA Modification
In some embodiments, the CRISPR-associated proteins described herein can be fused to a base-editing domain, such as ADAR1, ADAR2, APOBEC, or activation-induced cytidine deaminase (AID), and can be used to modify an RNA sequence (e.g., an mRNA). In some embodiments, the CRISPR-associated protein includes one or more mutations (e.g., in a catalytic domain), which renders the CRISPR-associated protein incapable of cleaving RNA.
In some embodiments, the CRISPR-associated proteins can be used with an RNA- binding fusion polypeptide comprising a base-editing domain (e.g., ADAR1, ADAR2, APOBEC, or AID) fused to an RNA-binding domain, such as MS2 (also known as MS2 coat protein), Qbeta (also known as Qbeta coat protein), or PP7 (also known as PP7 coat protein). The amino acid sequences of the RNA-binding domains MS2, Qbeta, and PP7 are provided below:
MS2 (MS2 coat protein)
MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIK VEVPKVATQTVGGVELPVAAWRSYLNMEL IPI FATNSDCELIVKAMQGLLKDGNPI PSAIA ANSGIY (SEQ ID NO: 171) Qbeta (Qbeta coat protein)
MAKLETVTLGNI GKDGKQT LVLNPRGVNPTNGVAS LS QAGAVPALEKRVTVSVS QPS RNRKN YKVQVKI QNPTACTANGS C DPSVT RQAYADVT FS FTQY ST DEERAFVRTELAALLAS PLL I D AI DQLNPAY ( S EQ I D NO : 17 2 )
PP7 (PP7 coat protein)
MS KT IVLSVGEATRTLTE I QSTADRQI FEEKVG PLVGRLRLTAS LRQNGAKTAYRVNLKLDQ ADWDCS SVCGEL PKVRYTQVWS HDVT IVANS TEAS RKS LY DLTKS LWQA S EDLWNLV PLGR ( S EQ I D NO : 155 )
In some embodiments, the RNA binding domain can bind to a specific sequence (e.g., an aptamer sequence) or secondary structure motifs on a crRNA of the system described herein (e.g., when the crRNA is in an effector-crRNA complex), thereby recruiting the RNA binding fusion polypeptide (which has a base-editing domain) to the effector complex. For example, in some embodiments, the CRISPR system includes a CRISPR associated protein, a crRNA having an aptamer sequence (e.g., an MS2 binding loop, a QBeta binding loop, or a PP7 binding loop), and a RNA-binding fusion polypeptide having a base-editing domain fused to an RNA-binding domain that specifically binds to the aptamer sequence. In this system, the CRISPR-associated protein forms a complex with the crRNA having the aptamer sequence. Further the RNA-binding fusion polypeptide binds to the crRNA (via the aptamer sequence) thereby forming a tripartite complex that can modify a target RNA.
Methods of using CRISPR systems for base editing are described, e.g., in
International Publication No. WO 2017/219027, which is incorporated herein by reference in its entirety, and in particular with respect to its discussion of RNA modification.
RNA Splicing
In some embodiments, an inactivated CRISPR-associated protein described herein (e.g., a CRISPR associated protein having one or more mutations in a catalytic domain) can be used to target and bind to specific splicing sites on RNA transcripts. Binding of the inactivated CRISPR-associated protein to the RNA may sterically inhibit interaction of the spliceosome with the transcript, enabling alteration in the frequency of generation of specific transcript isoforms. Methods of using CRISPR systems to alter splicing are described, e.g., in International Publication No. WO 2017/219027, which is incorporated herein by reference in its entirety, and in particular with respect to its discussion of RNA splicing. Therapeutic Applications
The CRISPR systems described herein can have various therapeutic applications. In some embodiments, the new CRISPR systems can be used to treat various diseases and disorders, e.g., genetic disorders (e.g., monogenetic diseases), diseases that can be treated by nuclease activity (e.g., Pcsk9 targeting, Duchenne Muscular Dystrophy (DMD), BCL1 la targeting), and various cancers, etc.
In some embodiments, the CRISPR systems described herein can be used to edit a target nucleic acid to modify the target nucleic acid (e.g., by inserting, deleting, or mutating one or more nucleic acid residues). For example, in some embodiments the CRISPR systems described herein comprise an exogenous donor template nucleic acid (e.g., a DNA molecule or a RNA molecule), which comprises a desirable nucleic acid sequence. Upon resolution of a cleavage event induced with the CRISPR system described herein, the molecular machinery of the cell will utilize the exogenous donor template nucleic acid in repairing and/or resolving the cleavage event. Alternatively, the molecular machinery of the cell can utilize an endogenous template in repairing and/or resolving the cleavage event. In some embodiments, the CRISPR systems described herein may be used to alter a target nucleic acid resulting in an insertion, a deletion, and/or a point mutation). In some embodiments, the insertion is a scarless insertion (i.e., the insertion of an intended nucleic acid sequence into a target nucleic acid resulting in no additional unintended nucleic acid sequence upon resolution of the cleavage event). Donor template nucleic acids may be double stranded or single stranded nucleic acid molecules (e.g., DNA or RNA). Methods of designing exogenous donor template nucleic acids are described, for example, in International Publication No. WO 2016/094874 Al, the entire contents of which are expressly incorporated herein by reference.
In one aspect, the CRISPR systems described herein can be used for treating a disease caused by overexpression of RNAs, toxic RNAs, and/or mutated RNAs (e.g., splicing defects or truncations). For example, expression of toxic RNAs may be associated with the formation of nuclear inclusions and late-onset degenerative changes in brain, heart, or skeletal muscle. In some embodiments, the disorder is myotonic dystrophy. In myotonic dystrophy, the main pathogenic effect of the toxic RNAs is to sequester binding proteins and compromise the regulation of alternative splicing (see, e.g., Osborne et al, "RNA-dominant diseases," Hum. Mol. Genet., 2009 Apr 15; 18(8): 1471-81). Myotonic dystrophy (dystrophia my otonica (DM)) is of particular interest to geneticists because it produces an extremely wide range of clinical features. The classical form of DM, which is now called DM type 1 (DM1), is caused by an expansion of CTG repeats in the 3 '-untranslated region (UTR) of DMPK, a gene encoding a cytosolic protein kinase. The CRISPR systems as described herein can target overexpressed RNA or toxic RNA, e.g., the DMPK gene or any of the mis-regulated alternative splicing in DM1 skeletal muscle, heart, or brain.
The CRISPR systems described herein can also target trans-acting mutations affecting RNA- dependent functions that cause various diseases such as, e.g., Prader Willi syndrome, Spinal muscular atrophy (SMA), and Dyskeratosis congenita. A list of diseases that can be treated using the CRISPR systems described herein is summarized in Cooper et al, "RNA and disease," Cell, 136.4 (2009): 777-793, and WO 2016/205764 Al, both of which are incorporated herein by reference in the entirety. Those of skill in this field will understand how to use the new CRISPR systems to treat these diseases.
The CRISPR systems described herein can also be used in the treatment of various tauopathies, including, e.g., primary and secondary tauopathies, such as primary age-related tauopathy (PART)/Neurofibrillary tangle (NFT)-predominant senile dementia (with NFTs similar to those seen in Alzheimer Disease (AD), but without plaques), dementia pugilistica (chronic traumatic encephalopathy), and progressive supranuclear palsy. A useful list of tauopathies and methods of treating these diseases are described, e.g., in WO 2016205764, which is incorporated herein by reference in its entirety.
The CRISPR systems described herein can also be used to target mutations disrupting the cis-acting splicing codes that can cause splicing defects and diseases. These diseases include, e.g., motor neuron degenerative disease that results from deletion of the SMNl gene (e.g., spinal muscular atrophy), Duchenne Muscular Dystrophy (DMD), frontotemporal dementia, and Parkinsonism linked to chromosome 17 (FTDP-17), and cystic fibrosis.
The CRISPR systems described herein can further be used for antiviral activity, in particular against RNA viruses. The CRISPR-associated proteins can target the viral RNAs using suitable RNA guides selected to target viral RNA sequences.
The CRISPR systems described herein can also be used to treat a cancer in a subject (e.g., a human subject). For example, the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule that is aberrant (e.g., comprises a point mutation or are alternatively-spliced) and found in cancer cells to induce cell death in the cancer cells (e.g., via apoptosis). Further, the CRISPR systems described herein can also be used to treat an infectious disease in a subject. For example, the CRISPR-associated proteins described herein can be programmed with crRNA targeting a RNA molecule expressed by an infectious agent (e.g., a bacteria, a virus, a parasite or a protozoan) in order to target and induce cell death in the infectious agent cell. The CRISPR systems may also be used to treat diseases where an intracellular infectious agent infects the cells of a host subject. By programming the CRISPR-associated protein to target a RNA molecule encoded by an infectious agent gene, cells infected with the infectious agent can be targeted and cell death induced.
Furthermore, in vitro RNA sensing assays can be used to detect specific RNA substrates. The CRISPR-associated proteins can be used for RNA-based sensing in living cells. Examples of applications are diagnostics by sensing of, for examples, disease-specific RNAs.
A detailed description of therapeutic applications of the CRISPR systems described herein can be found, e.g., in US 8795965, EP 3009511, WO 2016205764, and WO
2017070605; each of which is incorporated herein by reference in its entirety.
Delivery
Through this disclosure and the knowledge in the art, the CRISPR systems described herein, or components thereof, nucleic acid molecules thereof, and/or nucleic acid molecules encoding or providing components thereof, can be delivered by various delivery systems such as vectors, e.g., plasmids and viral delivery vectors (e.g., adeno-associated virus AAV vectors). The CRISPR-associated proteins and/or any of the RNAs (e.g., RNA guides) and/or accessory proteins can be delivered using suitable vectors, e.g., plasmids or viral vectors, such as adeno-associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or combinations thereof. The proteins and one or more crRNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors. For bacterial applications, the nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to the bacteria using a phage. Exemplary phages, include, but are not limited to, T4 phage, Mu, λ phage, T5 phage, T7 phage, T3 phage, Φ29, M13, MS2, Q , and ΦΧ174.
In some embodiments, the vectors, e.g., plasmids or viral vectors, are delivered to the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
In certain embodiments, the delivery is via adenoviruses, which can be at a single dose containing at least 1 x 105 particles (also referred to as particle units, pu) of
adenoviruses. In some embodiments, the dose preferably is at least about 1 x 106 particles, at least about 1 x 107 particles, at least about 1 x 108 particles, and at least about 1 x 109 particles of the adenoviruses. Exemplary delivery methods and the doses are described, e.g., in WO 2016205764 Al and U.S. Patent No. 8,454,972 B2, both of which are incorporated herein by reference in the entirety.
In some embodiments, the delivery is via a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, a modified AAV vector may be used for delivery. Modified AAV vectors can be based on one or more of several capside types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV 8.2. AAV9, AAV rhlO, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV 6) and pseudotyped AAV (e.g., AAV2/8, AAV2/5 and AAV2/6). Exemplary AAV vectors and techniques that may be used to produce rAAV particles are known in the art (see, e.g. , Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong ei al. (2012) J. Genet. Syndr. Gene Ther. SI : 008; West et al. (1987) Virology 160: 38-47 (1987); Tratschin et al. (1985) o/. Cell. Biol. 5: 3251-60); U.S. Patent Nos. 4,797,368 and 5,173,414; and International Publication Nos. WO 2015/054653 and WO 93/24641, each of which are incorporated by reference).
In some embodiments, the delivery is via plasmids. The dosage can be a sufficient number of plasmids to elicit a response. In some cases, suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg. Plasmids will generally include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR-associated proteins and/or an accessory protein, each operably linked to a promoter (e.g., the same promoter or a different promoter); (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmids can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on different vectors. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or a person skilled in the art.
In another embodiment, the delivery is via liposomes or lipofection formulations and the like, and can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Pat. Nos. 5,593,972; 5,589,466; and 5,580,859; each of which is incorporated herein by reference in its entirety.
In some embodiments, the delivery is via nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful in delivery RNA.
Further means of introducing one or more components of the new CRISPR systems to the cell is by using cell penetrating peptides (CPP). In some embodiments, a cell penetrating peptide is linked to the CRISPR-associated proteins. In some embodiments, the CRISPR- associated proteins and/or RNA guides are coupled to one or more CPPs to effectively transport them inside cells (e.g., plant protoplasts). In some embodiments, the CRISPR- associated proteins and/or RNA guides are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cell delivery.
CPPs are short peptides of fewer than 35 amino acids derived either from proteins or from chimeric sequences capable of transporting biomolecules across cell membrane in a receptor independent manner. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having proline- rich and anti-microbial sequences, and chimeric or bipartite peptides. Examples of CPPs include, e.g., Tat (which is a nuclear transcriptional activator protein required for viral replication by HIV type 1), penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide. CPPs and methods of using them are described, e.g., in Hallbrink et al, "Prediction of cell-penetrating peptides," Methods Mol. Biol., 2015;1324:39-58; Ramakrishna et al, "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA," Genome Res., 2014 Jun;24(6): 1020-7; and WO 2016205764 Al ; each of which is incorporated herein by reference in its entirety.
Various delivery methods for the CRISPR systems described herein are also described, e.g., in US 8795965, EP 3009511, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference in its entirety. Methods of Identifying CRISPR-Associated Protein Families
in one aspect, the disclosure relates to the use of computational methods and algorithms to search for and identify novel protein families that exhibit a strong cooccurrence pattern with certain other features within naturally occurring genome sequences. In certain embodiments, these computational methods are directed to identifying protein families that co-occur in close proximity to CRISPR arrays. However, the methods disclosed herein are useful in identifying proteins that naturally occur within close proximity to other features, both non-coding and protein-coding (for example, CRISPR Casl proteins). It should be understood that the methods and calculations described herein may be performed on one or more computing devices.
In some embodiments, a set of genomic sequences are obtained from genomic or metagenomie databases. The databases comprise short reads, contig level data, assembled scaffolds, or complete organisms. Likewise, the database may comprise genomic sequence data from prokaryotic organisms, or eukaryotic organisms, or may include data from metagenomie environmental samples. Exemplary database repositories include NCBI RefSeq, NCBI GenBank, NCBI Whole Genome Shotgun (WGS), and JGI integrated Microbial Genomes (IMG).
In some embodiments, a minimum size requirement is imposed to select genome sequence data of a specified minimum length. In certain exemplary embodiments, the minimum contig length may be 100 nucleotides, 500 nt, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 40 kb, or 50 kb.
In some embodiments, known or predicted proteins are extracted from the complete or a selected set of genome sequence data. In some embodiments, known or predicted proteins are taken from extracting coding sequence (CDS) annotations provided by the source database. In some embodiments, predicted proteins are determined by applying a
computational method to identify proteins from nucleotide sequences, in some embodiments, the GeneMark Suite is used to predict proteins from genome sequences. In some
embodiments, Prodigal is used to predict proteins from genome sequences. In some embodiments, multiple protein prediction algorithms may be used over the same set of sequence data with the resulting set of proteins de-duplicated.
In some embodiments, CRISPR arrays are identified from the genome sequence data. In some embodiments, PILER-CR is used to identity CRISPR arrays. In some embodiments, CRISPR Recognition Tool (CRT) is used to identify CR1SPR arrays. In some embodiments, multiple CRISPR array identification tools may be used over the same set of sequence data with the resulting set of CRISPR arrays de-duplicated.
In some embodiments, proteins in close proximity to CRISPR. arrays are identified. In some embodiments, proximity is defined as a nucleotide distance, and may be within 20 kb, 15 kb, or 5 kb. In some embodiments, proximity is defined as the number of open reading frames (ORFs) between a protein and a CRISPR array, and certain exemplar}' distances may be 10, 5, 4, 3, 2, 1, or 0 ORFs. The proteins identified as being within close proximity to a CRISPR array are then grouped into clusters of homologous proteins. In some embodiments, blastclust is used to form protein clusters. In certain other embodiments, mraseqs2 is used to form protein clusters.
To establish a pattern of strong co-occurrence between the members of a protein cluster with CRISPR arrays, a BLAST search of each member of the protein family may be performed over the complete set of known and predicted proteins previously compiled. In some embodiments, UBLAST or mmseqs2 may be used to search for similar proteins. In some embodiments, a search may be performed only for a representative subset of proteins in the family.
In some embodiments, the clusters of proteins within close proximity to CRISPR. arrays are ranked or filtered by a metric to determine co-occurrence. One exemplar}' metric is the ratio of the size of the protein cluster against the number of BLAST matches up to a certain E value threshold. In some embodiments, a constant E value threshold may be used. In other embodiments, the E value threshold may be determined by the most distant members of the protein cluster. In some embodiments, the global set of proteins is clustered and the cooccurrence metric is the ratio of the size of the CRISPR associated cluster against the size(s) of the containing global cluster(s).
In some embodiments, a manual review process is used to evaluate the potential functionality and the minimal set of components of an engineered system based on the naturally occurring locus structure of the proteins in the cluster. In some embodiments, a graphical representation of the protem cluster may assist in the manual review, and may contain information including pairwise sequence similarity, phylogenetic tree, source organisms / environments, and a graphical depiction of locus structures. In some
embodiments, the graphical depiction of locus structures may filter for nearby protein families that have a high representation. In some embodiments, representation may be calculated by the ratio of the number of related nearby proteins against the size(s) of the containing global cluster(s). In certain exemplary embodiments, the graphical representation of the protein cluster may contain a depiction of the CRISPR array structures of the naturally occurring loci, in some embodiments, the graphical representation of the protein cluster may contain a depiction of the number of conserved direct repeats versus the length of the putative CRISPR array, or the number of unique spacer sequences versus the length of the putative CRISPR array. In some embodiments, the graphical representation of the protem cluster may contain a depiction of various metrics of co-occurrence of the putative effector with CRISPR arrays predict new CRiSPR-Cas systems and identify their components.
The broad natural diversity of CRISPR-Cas defense systems contains a wide range of activity mechanisms and functional elements that can be harnessed for programmable biotechnologies. In a natural system, these mechanisms and parameters enable efficient defense against foreign DNA and viruses while providing self vs. non-self-discrimination to avoid self-targeting. In an engineered system, the same mechanisms and parameters also provide a di verse toolbox of molecular technologies and define the boundaries of the targeting space. For instance, systems Cas9 and Casl3a have canonical DNA and RNA endonuclease activity and their targeting spaces are defined by the proiospacer adjacent motif (PAM) on targeted DNA and protospacer flanking sites (PFS) on targeted RNA, respectively.
The methods described herein can be used to discover additional mechanisms and parameters within single submit Class 2 effector systems that can be more effectively harnessed for programmable biotechnologies.
Pooled-Screening
To efficiently validate the activity of the engineered novel CRISPR-Cas systems and simultaneously evaluate in an unbiased manner different activity mechanisms and functional parameters, a new pooled-screening approach was developed in E. coii. First, from the computational identification of the conserved protein and noncoding elements of the novel CRISPR-Cas system, these separate components were assembled into an engineered locus, which in one embodiment is on a single artificial expression vector based on the pET-28a+ backbone; in another embodiment, multiple compatible expression plasmids were used to recapitulate the engineered locus. To construct the vector, in one embodiment, DNA synthesis was used to assemble the components together: in another embodiment, molecular clonmg was used for assembly, in another embodiment, the proteins and noncodmg elements are transcribed on a single mRNA transcript, and different ribosomal binding sites are used to translate individual proteins.
Second, a library of unprocessed crRNAs consisting of the direct
repeat: : spacer: :direct repeat sequence was cloned into the engineered locus, in one embodiment, the spacers were targeting a second plasmid, pACYC 184, and the spacers were of the length found in the natural CRiSPR array. This crR A library was cloned into the vector backbone containing the proteins and noncoding elements (e.g. pET-28a+), and then subsequently transformed the library into E. coli along with the second target plasmid (e.g., pACYC184). It is important to have the plasmid(s) containing the engineered loci be on compatible origin(s) of replication with respect to the target plasmid to enable bacterial co- transformation. Consequently, each resulting E. coli cell contains no more than one targeting spacer.
Third, the E. coli were grown under antibiotic selection. In one embodiment, triple antibiotic selection is used: kanamycin for ensuring s uccessful transformation of the pET- 8 ! vector containing the engineered CRISPR-Cas effector system, and chloramphenicol and tetracycline for ensuring successful co-transformation of the pAC YCI 84 target vector. Since pACYC184 normally confers resistance to chloramphenicol and tetracycline, under antibiotic selection, positi ve activity of the novel CRISPR-Cas system targeting the plasmid will eliminate cells that actively express the proteins, noncoding elements, and specific active elements of the crRNA library. Using deep sequencing (e.g., next-generation sequencing), examining the population of surviving cells at a later time point compared to an earlier time point results in a depleted signal specifically for the active elements compared to the inactive crRNAs.
Since the pACYC184 plasmid contains a diverse set of features and sequences that may affect the activity of a CRISPR-Cas system, mapping the active crRNAs from the pooled screen onto pACYC184 provides patterns of activity that can be suggestive of different activity mechanisms and functional parameters in a broad, hypothesis-agnostic manner. In this way, the features required for reconstituting the novel CRISPR-Cas system in a heterologous prokaryotic species can be more comprehensively tested and studied.
The key advantages of the in vivo pooled-screen described herein include: (1) Versatility - engineered locus design allows multiple proteins and/or noncoding elements to be expressed; the library cloning strategy enables both transcriptional directions of the computationally predicted crRNA to be expressed;
(2) Comprehensive tests of activity mechanisms & functional parameters - Evaluates diverse interference mechanisms, including DNA or RNA cleavage; examines co-occurrence of features such as transcription, plasmid DNA replication; and flanking sequences for crRNA library can be used to reliably determine PAMs with complexity equivalence of 4N's;
(3) Sensitivity - pACYC 184 is a low copy plasmid, enabling high sensitivity for CRISPR-Cas activity since even modest interference rates can eliminate the antibiotic resistance encoded by the plasmid; and
(4) Efficiency - Optimized molecular biology steps to enable greater speed and throughput RNA-sequencing and protein expression samples can be directly harvested from the surviving cells in the screen.
The novel CRISPR-Cas families described herein were evaluated using this in vivo pooled-screen to evaluate their operational elements, mechanisms and parameters, as well as then ability to be active and reprogrammed in an engineered system outside of their natural cellular environment.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1 - Building an Expanded Database of CRISPR-Cas Systems, and Searching for Type VI-D RNA-Targeting Systems
We developed a computational pipeline to produce an expanded database of class 2 CRISPR-Cas systems from genomic and metagenomic sources. Genome and metagenome sequences were downloaded from NCBI (Benson et al, 2013; Pruitt et al, 2012), NCBI whole genome sequencing (WGS), and DOE JGI Integrated Microbial Genomes (Markowitz et al, 2012). Proteins were predicted (Meta-GeneMark (Zhu et al, 2010) using the standard model MetaGeneMark vl.mod, and Prodigal (Hyatt et al., 2010) in anon mode) on all contigs at least 5kb in length, and de-duplicated in favor of pre-existing annotations to construct a complete protein database. CRISPR arrays were identified and protein sequences for ORFs located within +/- lOkb from CRISPR arrays were grouped into CRISPR-proximal protein clusters. Clusters of fewer than 4 proteins, or comprising proteins from fewer than 3 contigs were discarded. Each of these remaining protein clusters were considered to be a putative effector of a CRISPR-Cas system. In addition to the CRISPR array and putative effector protein, many CRISPR-Cas systems also include additional proteins that enable adaptation, crRNA processing, and defense. Potential additional CRISPR-Cas system components associated with each of the predicted effectors were identified as clusters of protein-coding genes with high effector co-occurrence, and CRISPR enrichment or CRISPR representation of at least 15%.
Effector co-occurrence was calculated as the percentage of loci containing the effector that also contain the potential co-occurring protein. The high co-occurrence threshold was a function of the cohesiveness of the effector cluster (more homogenous clusters requiring a higher threshold). The CRISPR enrichment was calculated as follows: 1) Up to 20 unique proteins were sampled from each protein cluster, and UBLAST (Edgar, 2010) was used to generate a rank ordered list of proteins by E-value from the complete protein database, 2) An E-value threshold was imposed to recover at least 50% of the members of the cluster, and 3) CRISPR enrichment was calculated by dividing the number of CRISPR-proximal proteins below the E-value threshold by the total number of proteins below the threshold. CRISPR representation was calculated as the percentage of effector-proximal proteins in a CRISPR- proximal protein cluster. All clustering operations were performed using mmseqs2
(Steinegger and Soding, 2017).
This information was incorporated into a database of (predicted) CRISPR-Cas systems, each composed of: 1) a CRISPR array, 2) a putative effector, and optionally, 3) clusters of potential co-acting proteins. Aggregating and processing a collection of more than 10 Tb of prokaryotic genomic and metagenomic sequence data from multiple sources, our pipeline produced a database of 293,985 putative CRISPR-Cas systems. One important difference from previously reported computational pipelines (Shmakov et al, 2015, 2017a; Smargon et al., 2017) is that we perform minimal filtering (e.g., imposing a minimum size on putative effector) in the intermediate stages of the search in order to expand the range for potential discovery of novel CRISPR-Cas systems. As such, the resulting database of putative CRISPR-Cas loci includes all previously characterized class 2 CRISPR-Cas systems, but also contains a considerable amount of noise, such as degraded, non-functional CRISPR- Cas loci. For functional characterization of this database of candidate CRISPR-Cas systems, we constructed multiple sequence alignment for each family of putative effectors using MAFFT (Katoh and Standley, 2013) and conducted an HMM search using HMMer (Eddy, 2011) against protein family databases Pfam (Finn et al, 2014) and Uniprot (Bateman et al., 2017), as well as a BLASTN search of CRISPR spacer sequences against a reference set of phages. This analysis led to the detection of protein families corresponding to all previously identified class 2 CRISPR-Cas systems, indicating a minimal false negative rate. To identify novel class 2 CRISPR-Cas systems, features included above for the prediction of the functions of putative CRISPR-Cas systems were used to rank candidate families for follow-up functional evaluation.
Genomic Survey of Type VI-D RNA-Targeting CRISPR-Cas Systems
To expand the repertoire of Cas nucleases for RNA manipulation and sensing, we searched our database for type VI CRISPR-Cas systems with effector proteins containing two HEPN-domains each (2-HEPN proteins). In addition to the previously identified 2-HEPN proteins, Casl3a, Casl3b, and Casl3c, we detected a group of 2-HEPN proteins distantly related to Cas 13a (effectors of type VI-A), primarily in Eubacterium and Ruminococcus , which we denote Casl3d. The amino acid sequences of Casl3d proteins show less than 8% identity to the most similar Casl3a sequences; nevertheless, statistically significant sequence similarity between Casl3d and Casl3a can be demonstrated using PSI-BLAST initiated with a profile made from the multiple alignment of Casl3a (E-value = 0.002). This significant similarity is primarily due to the conservation of the HEPN domain sequences between Casl3a and Casl3d, whereas the remaining portions of the protein sequences in the two families are highly divergent; in particular, Cas 13d proteins lack a counterpart to the Helical -
1 domain of Casl3a (FIGs. 5A-C). Phylogenetic analysis of the Casl3 proteins clearly shows that Casl3a and Casl3d form strongly supported clades (FIGs. 4A-B).
Additionally, Casl3d effectors are notably smaller than previously characterized class
2 CRISPR effectors, with a median size of 928 aa. For comparison, this median size is 190 aa (17%) less than that of Casl3c, more than 200 aa (18%) less than that of Casl3b, and more than 300 aa (26%) less than that of Casl3a (FIG. 2B). Taken together, these lines of evidence suggest that this distinct group of class 2 CRISPR-Cas systems are best classified as Type VI- D, with the effector denoted Casl3d (FIG. 2A). We found that 77% of Casl3d genes occur adjacent to CRISPR arrays, and for 19%, the adaptation module (Casl and Cas2 genes) is present in the vicinity (FIG. 1), suggesting that many Type VI-D loci encode CRISPR-Cas systems that are active in both adaptation and interference. Phylogenetic analysis indicates that Casl proteins associated with Type VI-D are monophyletic and, in accord with previous observations on other type VI systems, are affiliated with the type II-A clade (FIG. 3). Thus, in the case of type VI, the adaptation module seems to have co-evolved with the effector module.
Spacer sequences from CRISPR arrays within 3kb of Casl3d effectors were extracted. In the case of multiple contigs containing the same Casl3d sequence (e.g., duplicated locus), only the contig containing the longest CRISPR array was used. Subsequent spacer analysis closely follows the method described previously (Shmakov et al., 2017b). Briefly, the resulting 198 spacers were de-duplicated by comparison of direct and reverse complement sequences, to produce a set of 182 unique spacers. A BLASTN (Camacho et al, 2009) search with the command line parameters -word size 7 -gapopen 5 -gapextend 2 -reward 1 - penalty -3 was performed for the unique spacer set against a database comprising the virus and prokaryotic sequences in NCBI. To identify prophage regions, (i) all ORFs within 3kb of prokaryotic matches were collected; (ii) a PSI-BLAST search was conducted against the proteins extracted from the virus part of NCBI, using the command line parameters -seg no - evalue 0.000001 -dbsize 20000000; and (iii) a spacer hit was classified as prophage if it overlapped with an ORF with a viral match, or if two or more ORFs with viral matches were identified within the neighborhood of the spacer hit.
The CRISPR arrays adjacent to Casl 3d genes contain 198 spacers total, of which 182 are unique. A BLASTN search of the unique spacer sequences against a database comprising known phages and NCBI prokaryotic sequences revealed 7 spacers with significant hits (defined as E-value < 0.0001, alignment length at least 24, 0 gaps, and no more than one mismatch). One spacer, from Ruminococcus flavefaciens FD-1, showed significant matches against the Arthrobacter dsDNA phage Gordon (alignment length=28, 1 mismatch) and against a putative prophage region in an uncultured Flavonifractor sequence (alignment length=24, 0 mismatches). A different spacer, from a gut metagenome sequence, resulted in a significant match against a putative prophage region in Bacillus soli (alignment length=24, 0 mismatches). The remaining five spacer matches targeted ORFs in prokaryotic sequences, but were not classified as being in prophage regions. The presence of spacers homologous to DNA phage genomic sequences in an RNA-targeting CRISPR-Cas system might appear unexpected but is in line with similar observations on type VI-A and type VI-B systems (Smargon et al, 2017). Presumably, type VI systems abrogate the reproduction of DNA phages by cleaving phage mRNAs, but the mechanistic details of the antivirus activity of these systems remain to be characterized experimentally.
Examination of the additional genes in the vicinity of Casl3d led to the identification in most of the VI-D loci of potential accessory proteins containing WYL domains (so denoted after three amino acids that were conserved in the originally identified group of these domains) and additionally, ribbon-helix-helix (RHH) DNA-binding domains (FIG. 6).
For phylogenetic analysis of these Casl3d-associated WYL-domain containing proteins, we compiled a data set of WYL proteins. In addition to automatically identified WYL proteins, we used PSI-BLAST (Altschul et al., 1997) to search over a local set of NCBI sourced proteins using RspWYLl as a query. The results with E-value 0.01 or lower were added to the set of WYL proteins. Proteins smaller than 150 aa were discarded from the data set, and UCLUST (Edgar, 2010) with identity threshold 0.90 was used to obtain a non- redundant set. We then added all WYL proteins identified in the vicinity of Casl3d genes to form a set of 3908 WYL sequences for phylogenetic analysis. Multiple alignment and phylogeny of protein sequences were constructed as described previously (Peters et al., 2017).
Briefly, the sequences were clustered by similarity, and for each cluster, a multiple alignment was built using MUSCLE (Edgar, 2004). Alignments were combined into larger aligned clusters by HHalign (Yu et al, 2015) if the resulting score between the two alignments was higher than the threshold; otherwise, the scores were recorded in a similarity matrix. The matrix was used to reconstruct a UPGMA tree. For each cluster, the alignment was filtered as follows: the alignment positions with the gap character fraction values of 0.5 and homogeneity values of 0.1 or less were removed. The remaining positions were used for tree reconstruction using FastTree with the WAG evolutionary model and the discrete gamma model with 20 rate categories. The same program was used to compute SH (Shimodaira- Hasegawa)-like node support values
The WYL-domain proteins contained in Type VI-D loci fall into six strongly supported branches of the broader phylogenetic tree of WYL-domain proteins. The branch we denote WYL1 is a single WYL-domain protein associated primarily with Ruminococcus . Multiple sequence alignment of WYL 1 shows an N-terminal RHH domain, as well as a pattern of primarily hydrophobic conserved residues, including an invariant tyrosine-leucine doublet corresponding to the original WYL motif (FIG. 7). Other VI-D loci contain duplicated genes encoding WYL-domain proteins, as in Ruminococcus flavefaciens, or a fusion of two WYL-domain proteins, as in Eubacterium sp. Although a substantial majority of the VI-D loci encode WYL-domain proteins, phylogenetic analysis indicates that these CRISPR-associated WYL proteins are scattered among different branches of the WYL family tree, i.e., are polyphyletic. Thus, the VI-D CRISPR-Cas systems appear to have acquired WYL-domain proteins on several independent occasions, suggesting a role for this protein in modulating the CRISPR-Cas function.
Exemplary Type VI-D CRISPR-Cas effector proteins are provided in TABLES 1 and 2 (e.g., SEQ ID NOs. 1-31, and 200-350). Exemplary Type VI-D CRISPR-Cas direct repeat sequences are provided in TABLE 3 (e.g., SEQ ID NOs 32-49, 52-77, 351-589). Exemplary Type VI-D CRISPR-Cas associated WYL accessory proteins are provided in TABLES 1, 4, 5, and 6. In some embodiments, a Type VI-D CRISPR-Cas effector protein comprises an exemplary motif provided in TABLE 7 (e.g., SEQ ID NOs: 94-98, 672 and 673).
Table 1. Representative CaslSd Effector and WYLl Accessory Proteins
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Table 2. Amino Acid Sequences of CaslSd Effector Proteins
>LARF01000048_8
[Ruminococcus sp . N15.MGS-57]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKGSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLSIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 2)
>WP_005358205.1
[ [Eubacterium] siraeum DSM 15702]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVPKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGRKKDEPEQSVPTDMLCLKPTLEKKFF GKEFDDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKKLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIESDKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIKFENFSDAGNYQKANVTSRTSEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSFAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIKEIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDEQK (SEQ ID NO: 1)
>3300010266 I Ga0129314_1001134_19
[animal-digestive system-orangutan individual fecal]
MGKKIHARDLREQRKNDRTTKFAEQNKKREAQMAVQKKDAAVSAKSVSSVSSKKGNVTKSMAKAAGVKSVFAVGKNTVYMTSFG RGNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAITDNPLRRFNGGKKDKPEQSVPADMLCLKPTLEKKF FGKEFDDNIHIQLIYNILDIEKILAVYSTNAVYALNNTIADENNENWDLFANFSTDNTYGELINAATYKESTDDVSTDDEKRRE AEKKKREAKIAEKILADYEKFRKNNRLAYFADAFYIEKNKSKSKSQNKAEGIKRGKKEIYSILALIAKLRHWCVHSEDGRAEF LYKLDELEDDFKNVLDWYNRPVEEINDDFVERNKVNIQILHSKCENSDIAELTRSYYEFLITKKYKNMGFSIKKLREIILEGT EYNDNKYDTVRNKLYQMVDFILYRGYINENSERAEALVNALRSTLNEDDKTKLYSSEAAFLKRKYMKI IREVTDSLDVKKLKEL KKNAFTIPDNELRKCFISYADSVSEFTKLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLERTFTDEYSFFEGSTK YLAELIELNSFVKSCSFDMSAKRPMYRDALDILGIESDKSEDDIKRMIDNILQVDANGKKLPNKNHGLRNFIASNWESNRFEY LVRYGNPKKIRETAKCKPAVRFVLNEIPDAQIERYYKAYYLDEKSLCLANMQRDKLAGVIADIKFDDFSDAGSYQKANATSTKI TSEAEIKRKNQAIIRLYLTVMYIMLKNLWWARYVIAFHCLERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGIIKTEF DKSLAENAANRYLRNARWYKLILDNLKMSERAWNEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADNGG STGDYIGKLEEHKTYCKDFVKAYCTPFGYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 3)
>3300006226 I Ga0099364_10024192_5
[ arthropoda-digestive system-cubitermes and nasutitermes termite gut]
MSQSTKTKAKRMGVKSVLAHGKDEKGHIKLAITAFGKGNKAELAIQTDEKGSNLAKTYKERNITANKIVSEGIQTSGTIAGEGH ATFLNNPAEHVGTDYLKLKETLEMEFFGKSFPGDSVRIQI IHQILDIQKLLGIYITDI IYCINNLRDETHLDHESDIVGLSMSN TNWLALNQMRPYFGFFGEAFRPVGDDKVKEITLSDEVRKNIEKIIALEEQKRNPSTPRFKQENINLEIENAMGKFKSKDAFET AKKKYNRIVADETNAKTLRILGAMRQITAHFKDQATLFMSDVELPKILKKEFSKADWQTVEDYYAKLVDRINEGFCKNAATNVH FLTELLPEESKKQLTEDYFRFAILKEGKNLGVNMKRLREVMFALFVPELTAPETKKRYDSYRAKIYGLTDFLLFKHIHNTKQLE EWVAVLRETSNEDAKENLYDEFARTAWNTVGDSAKQLIENMQSYFTKKEKEITKTAQPVLSTSSIAHTSKKITQFSSFAKLLAF LCNFWEGKEINELLSAYIHKFENIQEFINLLEKLEGKKPQFTENYALFNEAAGQRAGEIAQNLRILASIGKMKPDLGDAKRQLY KAAIEMLGIDTEEYISDEWLEPNMLLAQPPKEPKKDNEKYRKEPHKYSYEKDMETYRKKLREYEETWRSLIDYEYLMPETNPFR NFVAKQVIESRRFMYLVRYTKPKTVRALMSNRAIVHYVLSRIADIQDHHMTESQIDRYYQNLPQYNEQQHKNVSLETKIDALAD YLCKYTFEKNVLKQKNGIVLNTKSATKNVEIEHLKALTGLYLTVAYIAVKNLVKANARYYIAFSIFERDYALFEKKLGKDTLEK YVKPFKYIDKGEEKEGKNNFFALTEYLLDKDNSLRYQWNNDLSDEENKQALRKHLDKKEIRSQRHFSQYWLDIFARQIENAKKT SESGYLLTAARNCALHLNVLTALPEFVGEFRKTGDKMTSYFELYHFLLQKLMLAEAGLNLDEYRERIDTYQTACKDLINITYVS LGYNLPRYKNLTCEPLFDEESATGKERQTRLDEKSKEKKQRKGGQK (SEQ ID NO: 4) >NZ_NFLV01000009_111
[ Eubacterium sp. Anil]
MSKKQRPKDIRKRQEEEKREKYKKQEELRKKQEELRKEQEQRREDQKELEKIKKEVGEEGEKKKSRAKALGLKSTFILDRDEQK VLMTSFGQGNKAVRDKYI IGDKVSDINDDRKNKKAALLVEVCGKSFNI SKKENDDCDPVKWNPVVSRNKKDDDLIHCRKKLEE LYFGEQFKDNIHIQLIYNILDIEKILAVQVNNIVFALNNLLSWSGEEKFDLIGYLGWDTYEKFRDAKGKRKGLYEKFSTLIEK KRMRYFGSTFYPLNEKGEEITSNDKKEWEQFEKKCYHLLAVLGMMRQATAHGDSKRRAEIYKLGKEFDKSEARGCRQEARKELD DLYRKKIHEMNQSFLKNSKRDILMLFRIYDAESKEAKRKLAQEYYEFIMLKSYKNTGFSIKHLRETVIDKMDEDIKEKIKDDKY NPIRRKLYRIMDFVIYQYYQESEQQEEAMELVRKLRNAETKVEKELTYRKEAEKLKEELEKI IRNSILSVCDRILAEMNEKRHK KVNQESSDTDSEEPLDPEISEGITFIKETAHSFSEMIYLLTVFLDGKEINILLTQLIHCFDNISSFMDTMKEENLLTKLKEDYE IFEESKEI SKELRI INSFARMTEPVPKTEKTMFIDAAQILGYSNDEKELEGYVDALLDTKNKTKDKERKGFEKYIWNNVIKSTR FRYLVRYADPKKVRAFAANKKWAFVLKDI PDEQIKAYYNSCFSQNSDSSSNMSIAFQDGDSNKKGTSVHDMMRKALTEKITGL NFGDFEEESKKGIRREESDKNI IRLYLTVLYLVQKNLIYVNSRYFLAFHCAERDEVLYNGETIDNNKEKGSEKDWKKFAKEFII EHPPKKKVKDYLAKNFEYSNKWSLRVFRNSVQHLNVIRDAYKYIKCIDDNKDVQSYFALYHYLVQRYI SEMAENLTDKGELSEG RLQYYLSQVENYRTYCKDFVKALNVPFAYNLPRYKNLSIDELFDRNNYLPNKAKKWISEKKENGEYVMEDCGNKGAGQVENA
(SEQ ID NO: 5)
>NFIR01000008_78
[Eubacterium sp. An3]
MAKKLRPKELREKRRMAEKEEHKKQEKLRKEQEELRKKQEKQREDQKELEKIKKEEGGEGEKKKSGAKALGLKSTFILDRDEQK MLMTSFGRGNKAVRDKYI IGDKVSDIDDSWENKKAALSVEVCGKSFNI SKKENDDCEPVKWNPVLSGNKKDDDLIHCRKNLEE MYFGQQFKDNIHIQLIYNILDIEKILAVQINNIVFILNNLLRWSGEEEFDLIGSLGWHTYEEFRGRNKNYGKFSELIKQSQMR YFGSTFCLFNENEERITSENKKEWKRFEKKCYHLLAVLGMMRQATAHGDSKRRAEIYKLGKEFDRLEARGCRPEARKELDELYK KKIHEMNQGFLKNSKSDILMLFRIYNAESKEAKRKLAQEYYEFIMLKSYKNTGFSIKHLRETMIDKMDEDKKEKLKDDKYNPIR RKIYRIMDFMIYQYYQEPEHQEEAEELVRKLRNAEIEAKKELAYRKEAEKLKKELEKI IFNSVLPSCDRILSEMDERRNKKVNQ ESSDTDKEEPLDSEIAEGITFIKETAHSFSEMIYLLTVFLDGKEINILLTQLIHCFDNISSFMDTMEEENLLTKLKEDYEIFEE SKEI SRELRI INSFARMTEPVPKTERIMFIEAAQILGYSNGEKELEGYVDALLDTKNKTNDKKKKGFVRYIWNNVIKSTRFRYL VRYADPKKVRAFAANKKWAFVLKDI PDDQIRAYYNSCFRQNSDSSSNNSNASWDADSNKRDISVSDMRKALTEKITGLNFGDF EEESKKGIRKEESDKNIIRLYLTVLYLVQKNLIYWSRYFLAFHCAERDEMLYNGETIDNNKEKGSEKDWRKFAKQFIMEHSPK KKVKDYLAKNFEYSNKWSLKEFRNSVQHLNVIRDAHKYIKYINDNKDVQSYFALYHYLVQRYISERAANRTDKESLSEGRLQYY LSQVKEYRTYCKDFVKALNVPFAYNLPRYKNLSIDELFDRNNYLPNKAKKWI PEKKENGEYVMEDCGNKDAGQVENA (SEQ ID NO: 6)
>CDYS01033339_14
[gut metagenorae]
MEREVKKPPKKSLAKAAGLKSTFVISPQEKELAMTAFGRGNDALLQKRIVDGWRDVAGEKQQFQVQRQDESRFRLQNSRLADR TVTADDPLHRAETPRRQPLGAGMDQLRRKAILEQKYFGRTFDDNIHIQLIYNILDIHKMLAVPANHIVHTLNLLGGYGETDFVG MLPAGLPYDKLRWKKKNGDTVDIKADIAAYAKRPQLAYLGAAFYDVTPGKSKRDAARGRVKREQDVYTILSLMSLLRQFCAHD SVRIWGQNTPAALYGLQALPQDMKDLLDDGWRRALGGVNDHFLDTNKVNLLTLFEYYGAETKQERVALTQDFYRFWLKEQKNM GFSLRRLREELLKLPDAAYLTGQEYDSVRQKLYMLLDFLLCRLYAQERADRCEELVSALRCALSDEEKDAVYQAEAAALWQALG DTLRRELLPLLKGKKLQDKDKKKLDELGLSRDVLDGVLFRPAQQGSPANADYFCRLMHLSTWFMDGKEINTLLTTLISKLENID SLRSVLESMGLAYSFVPAYAMFDHSRYIAGQLRVWNIARMRKPAIGAKREMYRAAWLLGVDSPEAAAAITDDLLQIDPETGK VRPRGDSARDTGLRNFVANNWESRRFTYLLRYMTPEQARVLAQNEKLIAFVLSTVPSAQLERYCRTCGREDITGRPAQIRYLT AQIMGVRYESFTDVEQRGRGDNPKKERYKALIGLYLTVLYLAVKNMWCNARYVIAFYCRDRDTALYQKEVCWYDLEEDKKSGK QRQVEDYTALTRYFVSQGYLNRHACGYLRSNMNGISNGLLAAYRNAVDHLNVIPPLGSLCRDIGRVDSYFALYHYAVQQYLNGR YYRKTPREQELFAAMAQHRTWCSDLVKALNTPFGYNLARYKNLSIDGLFDREGDHWREDGEKPAE (SEQ ID NO: 7)
>CDYU01004315_2
[gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEIKNNAVPAIAAMPAAEAAAPAVEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKRGNESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDF IEGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 8) >CDYX01024884_4
[gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDDNDYNKTQLSSKDNSNIELGNWEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRDTLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKIMDFLLFCNYY RNDVVAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMF RDALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEFPDMNSSLEVKRSELARMIKNICFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCL ERDFGLYKEIVSELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIRAVDSYFSIYHYVMQRCITKRGNDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 9)
>CDTW01032418_55
[gut metagenorae]
MEREVKKPPKKSLAKAAGLKSTFVISPQEKELAMTAFGRGNDALLQKRIVDGWRDVAGEKQQFQVQRQDESRFRLQNSRLADR TVTADDPLHRAETPRRQPLGAGMDQLRRKAILEQKYFGRTFDDNIHIQLIYNILDIHKMLAVPANHIVHTLNLLGGYGETDFVG MLPAGLPYDKLRWKKKNGDTVDIKADIAAYAKRPQLAYLGAAFYDVTPGKSKRDAARGRVKREQDVYAILSLMSLLRQFCAHD SVRIWGQNTTAALYHLQALPQDMKDLLDDGWRRALGGVNDHFLDTNKVNLLTLFEYYGAETKQARVALTQDFYRFWLKEQKNM GFSLRRLREELLKLPDAAYLTGQEYDSVRQKLYMLLDFLLCRLYAQERADRCEELVSALRCALSDEEKDTVYQAEAAALWQALG DTLRRKLLPLLKGKKLQDKDKKKSDELGLSRDVLDGVLFRPAQQGSPANADYFCRLMHLSTWFMDGKEINTLLTTLISKLENID SLRSVLESMGLAYSFVPAYAMFDHSRYIAGQLRVWNIARMRKPAIGAKREMYRAAWLLGVDSPEAAAAITDDLLQIDPETGK VRPRSDSARDTGLRNFIANNWESRRFTYLLRYMTPEQARVLAQNEKLIAFVLSTVPDTQLERYCRTCGREDITGRPAQIRYLT AQIMGVRYESFTDVEQRGRGDNPKKERYKALIGLYLTVLYLAVKNMWCNARYVIAFYCRDRDTALYQKEVCWYDLEEDKKSGK QRQVEDYTALTRYFVSQGYLNRHACGYLRSNMNGISNSLLTAYRNAVDHLNAIPPLGSLCRDIGRVDSYFALYHYAVQQYLNGR YYRKTPREQELFAAMAQHRTWCSDLVKALNTPFGYNLARYKNLSIDGLFDREGDHWREDGEKPAE (SEQ ID NO: 10)
>CDZT01047721_3
[gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLNYLVDERFDSIN KGFIQGNKWISLLIDMMKDDYEADDIIHLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFC NYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLY FSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKL TMFRDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQ IERYYKSCVEFPDMNSSLKVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAI HCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWR ELKEYIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNE YLTEK (SEQ ID NO: 11)
>ODXP01000624_4
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDSSNIELRGVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESHDDFMGYLSAKNTYDVFTNPNGSTLSDDKKKNIRKSLRKFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKI IDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 12)
>ODKA01005851_3
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSEDSSNIELCGVNKWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTNALEAYKKRVYYMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNYYRN DVVAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVENLWWARYVIAIHCLER DFGLYKEI ISELASKNLKNDYRILSQTLCELCDNCDESPNLFLKKNERLRKCVEVDINNADSNMTRKYRNCIAHLTWRELNKY IKDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 13)
>OGPQ01001037_3
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVDEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGGDESHDDIMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRASEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV IAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSMGAKRRELAKMIKSISFEDFKDVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELNKYIK DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKINYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 14)
>CDZK01015063_14
[gut metagenorae]
MFMAKKNKMKPRERREAQKKARQLKAAEINNNAVPAIAAMHAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAV LEYEVDNNDYNQTQLSSKDNSNIELCGVTKWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDD NIHIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKT KRLGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIYPEYRDTLDYLVEERLKSINKDFIQ GNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND WAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKKYI GDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 15)
>3300001598 | EMG_10000232_1
[mammals-digestive system-asian elephant fecal-elephas maximus]
MYNIDKLWLTHILFVSLTAGKKNETILEQEINKDSNKKNILVNPTKFDANIKEVRMVSIKPEKYNETVWNPYYVKDGQWGQD YLGIKDKLEDTFFGKTYDDNIHIQIAYKLLDIRKIMGMSVGSAVFSLNNLQQRPVGENPNDIVGQIKTDTSFDEIPDNYAKADK DFIDILLDYTRYFDNVFEKQSI SVDDKTKDILNNLKECETVSVKTVGTIDRINKNDPNNNNYTI FKIGGLKIKLKGILSNVDVG TKLNIEGQIRRNNDYRDKKGKLCRSYSLLTGAKYSI SHEVYNPDTYTFNYDILRLVSYLRQAWHNNNDDYIDWLYSIDNKKET KDILNAANKVFESQLEAFNKDFNANAQKNVYMIASVLNDSPKTMFKEEIKDIYEKYYNFVLFKENRNVGINLRNIRNNIFYEDI KPNYDEKELSRERAKINTLLDYFIYQDFNNNEKLAEDVIARLQPTKQEVDKVQVYADVTKEFKVRNPKLVDRILSTVKNTIEAK IENFIPDNCVPSSSIKVSSLAKYVYVLAKFLDTKEVNNLLTSLINSFENIGSLVKVLKDEKGYSIYKDRFALLNQKNPFDLAND FILVKNLATMKTKLAKANVKDVKNKVGKRLYCSAINLFKDKNDEVILDNQEFEDIMSEFSSNVGNKKNRRGTAGSKIRNFLINN VIDSRRFYFIIKYYDTRRCHEIIQNENLVRFILGREDMPTDQLIRYYKTITGNECNNRNQIIDTLVKKLKEVSFRKLLLKGERL KEIGNDQDNQEVESLKSLIGLYLTICYLIVKGIVNVNSVYLLAWSAYERDMYYLYNEDMEDKNTNHDYLKAATDFYNNKSCYQK RHKYLIKDIEEARQNSNNLNYKDYRNKVCHYNICTSFMDYANNIGKVSCYFDIYNYCFQRYFAKKNDNLSTLLDTYNCYNKDYL KLLNMPFAYNMARYKNLTIADLFNDKYPSENKEATASND (SEQ ID NO: 16)
>3300001598 | EMG_10003641_1
[mammals-digestive system-asian elephant fecal-elephas maximus]
MEETKVTKETTIEKQSTKRHKQKSKKTATKMSGLKSALVINNHEMLLTSFGKGNNAIAEKRYILDGDIETINNKNKKFDANNDS KVWIKGISNPNGQLTNPLFDQSPTAIQPNRTSGNDMIGIRRMLERKYFVHNEENKEFQDNIRIQIAYCILDIEKILMPHINNI CFEINNMLRLEGYQEDSFMGSFNLYKPYDAFIATTDDKESSRRDNFAKLMTSKQVRYLGNALYSDSLSNLTKDEILDGKRSKEL KKYYQELCLLGMVRQSMIHSNQFNSSIYTLDSSYDSTMNTAELLGKGDDSSLVALATDARVEARAILDEIYKKGVDSINNSFLS NSINDLENLFKIYKCDSSEKKTELIKQYYDFCIRKPQMNMGFSITTIREGMFTRCSEANTLLLCDEGSTVKLNVHDTMKSKFYK NLDFMIYKYYKYENPEKGEKLIEDLRSKIKGKKKEDEDKKQRYAEESACILKAKRDIIKKDLTEAANKDLFADLVKSNKNEKQK FKNEYEELLKPFMI PVKVDYFSELIYLVTRFLSGKEINDLLTQLINKFENIAAFIRMYQNDQGKLEFTANYKMFEIDPQKDI PK DGKRVLSGSAKIAYYLRTINYIARMESFEIKSDKTAINDAISLLGYNSNEHRDEFITYTMAKHWDKYQNTDYQKIVKDFLSAN KTLDCKSKNMQAFVSELKNAHLSENYEQREKEIYELADTNLPAYFSEEDKEKLARYIVHSDGTYKKFLKESFYAIEELPNEGFR NFISNNVINSRRFNYIMRFCNPEKIANIGKNKVLISFALSSLAEKTDMIAKYYRVFCDRIDDQKTMEDYLWKLTKISYTEFLN WQKANAEKNKEKDRSQKLIGLYITLLYEIVKNLWINSRYNIAFQRCDNDSIMILQGQYDERAVQESKLTKKFISNQKLNSYS CRYLTHNI SQLDRCNDFIRQYRNKVAHLEWSNIDEYLSGIKHIESYYALYHYLMQKCLLKNYRIEDHSQNEYKNLNDFSSKLD KHGTYVKDFVKALNVPFGYNLPRYKNLSIDELFDRNKLKTGGTIEMKGE (SEQ ID NO: 17)
>3300018475 I GaOl 87907_10006632_17
[mammals-digestive system- feces]
MKERIDMIEKKKSYAKGMGLKSTLVSDSKVYMTSFGNGNDARLEKVVENNAI SCLVDKKEAFVAEITDKNAGYKIINKKFGHPK GYDWANNPLYTGPVQQDMLGLKETLEKRYFGSSVSGNDNICIQVIHNILDIEKILAEYITNAAYAVNNIAGLDKDIIGFGKFS TVYTFDEFAEPDRHKERFIKDGKLDTKLINQLKNQYDEFDAFLDDTRFGYFGKAFFCKEGDKYLNKQDNERYHILALLSGLRNW WHNNEVESKIDRKWLYNLDKNLDKEYITTLDYMYSDIADELTKSFSKNSAANVNYIAEILNIDSKTFAEQYFRFSIMKEQKNL GFTLTKLRECMLDREELSDIRDNHKVFDSIRSKLYTMMDFVIYRYYIEEAKKIENENKTLSDDKKKLSEKDI FI ISLRGSFSEE QKDKLYSDEAERLWAKLGKLMLEIKKFRGQMTRDYKKSDTPTLNRILPESEDVSTFSKLMYALTMFLDGKEINELLTTLINKFD NIQSMLKIMPLIGVNAKFSSDYAFFNNSEKIADELKLIKSFARMGEPVANAKRDMMIDAIKILGTDLDDNEIKKLADSFFKDSN GKLLHKGKHGMRNFIINNWNNKRFHYIIRYGDPAHLHEIAKNEWVRFVLGRIADIQKKQGKGGKNQIDRYYEICIGNGYGKS VSEKIDALTKVI INMNYDQFEAKRKVIENTGRDNAEREKYKKII SLYLTVIYQILKNLWWSRYVIGFHCVERDAQLYKEKGY DINTNNLESKGFTSVTKLCVGIADDDPVKYKNVEIELKERALASFDALEKENPELYEKYNMYSEKQKEAELEKQINREKAKTAL NAHLRNTKWNVI IRENIRNTEKDACKQFRNKADHLEVARYAYKYINDI SEWSYFQLYHYIMQRII IDSSGNNANGMIKKYYES VI SDKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDKNEAAKYDKIKKKVAVR (SEQ ID NO: 18)
>3300018475 I GaOl 87907_10006632_17
[mammals-digestive system- feces]
MIEKKKSYAKGMGLKSTLVSDSKVYMTSFGNGNDARLEKVVENNAI SCLVDKKEAFVAEITDKNAGYKIINKKFGHPKGYDVVA NNPLYTGPVQQDMLGLKETLEKRYFGSSVSGNDNICIQVIHNILDIEKILAEYITNAAYAVNNIAGLDKDIIGFGKFSTVYTFD EFAEPDRHKERFIKDGKLDTKLINQLKNQYDEFDAFLDDTRFGYFGKAFFCKEGDKYLNKQDNERYHILALLSGLRNWWHNNE VESKIDRKWLYNLDKNLDKEYITTLDYMYSDIADELTKSFSKNSAANVNYIAEILNIDSKTFAEQYFRFSIMKEQKNLGFTLTK LRECMLDREELSDIRDNHKVFDSIRSKLYTMMDFVIYRYYIEEAKKIENENKTLSDDKKKLSEKDI FI ISLRGSFSEEQKDKLY SDEAERLWAKLGKLMLEIKKFRGQMTRDYKKSDTPTLNRILPESEDVSTFSKLMYALTMFLDGKEINELLTTLINKFDNIQSML KIMPLIGVNAKFSSDYAFFNNSEKIADELKLIKSFARMGEPVANAKRDMMIDAIKILGTDLDDNEIKKLADSFFKDSNGKLLHK GKHGMRNFIINNVVNNKRFHYI IRYGDPAHLHEIAKNEVWRFVLGRIADIQKKQGKGGKNQIDRYYEICIGNGYGKSVSEKID ALTKVIINMNYDQFEAKRKVIENTGRDNAEREKYKKII SLYLTVIYQILKNLVNVNSRYVIGFHCVERDAQLYKEKGYDINTNN LESKGFTSVTKLCVGIADDDPVKYKNVEIELKERALASFDALEKENPELYEKYNMYSEKQKEAELEKQINREKAKTALNAHLRN TKWNVI IRENIRNTEKDACKQFRNKADHLEVARYAYKYINDI SEWSYFQLYHYIMQRII IDSSGNNANGMIKKYYESVI SDKK YNDRLLKLLCVPFGYCIPRFKNLSIEALFDKNEAAKYDKIKKKVAVR (SEQ ID NO: 19)
>3300018494 I GaOl 87911_10069260_3
[mammals-digestive system- feces]
MSTKKRFRYSVAAKAAGLKSSLAVDTDRTVMTSFGHGNAAILEKEIVDGEISVLNIENPAFDAVINDKKYALTGHHAGVHALVD QPQNRSDAVHIRGALEKKYFGDTFADNIHVQIAYNILDITKILTVYANNWYALNNLVHADDDTQADELDSLGNFSAGTSYAKS KSKSKSKQQDFVELFIKKKEIHGYFGDTFAFLDKRIADADKEKQVYAMLACLGSLRQACSHYRIRYSVNGKNVDADADTWLFSS AQLDQTDPLFSEMLNRIYSHKIKTVNQNFFENNRKANFPILKKMYPETTLKVLMNEYYDFSIRKGYKNFGFSIKSLREALLSPQ YESLIGVQIKDNKEYDTVRSKLYQLFDFALTRYFNQHPDMVDAFWELRSLAKDEDAKNAVYEKYAKAVWNDVKQPIAVMLSYM NGSAIKNIKAFELKPDQKELNGIMNSNALDVPHFCKLVYFLTRFLDGKEINDLLTTLVNKFDNIHSFNQVLTALGLSASYEADY KI FEDSGRWEYLREINSFARMTVDMEKIKRSAYKKALLILGSSKYSDEDLDARVDEMLGVDYNQNGEKIKVRVDTGFRNFIAN NWESSRFHYLIRYCHPRKIRNLAGNAALIEYQLRRLPELQILRYYEACTEPIKRTARTMDEKIGTLIDLIVKMDFSQFEDVQQ NDRVRVFSDAEKKEKIRKMREKQRYQSI ISLYLTMLYLIVKNLVNINARYVMAFQAWERDNYLLLQLSGKEAEAEYLNLTRHFI EPLDGAKPYLKKRPVEYLKKDMAMVGNSSIRHFRNATVHLNVIMEAHRYTKDIKYIGSYYALYHYILQRHLLDKIEEDSYAEKT VSEKLWESQI SQYGTYSKDFVKALCCPFGYNLPRFKNLSIEQLFDRNESKEITDATAPRQ (SEQ ID NO: 20)
>3300018493 I GaOl 87909_10030832_9
[mammals-digestive system- feces]
MAKKKKAKQRREEQEAARMNKIQSAVKAKAETAPAVSSAFVEKRKDKQSKKTFAKASGLKSTLAVDNSAVMTVFGRGNEAKLDH RINADLQSESLHPQAALKNVHAPNKQKIHFIGRMQDMNLTADHPLHSHDGERAVGADLLCAKDKLEQLYFGRTFNDNIHIQLIY QILDIQKILALHANNI IFALDNLLHKKNDELSDDFVGMGRMRATIGYDAFRNSTNQKVQETYREFQEFVRRKELLYFGSAFYNG DTRRDEKVIYHILSLAASVRQFCFHNDYTSDDGKGFIKADWMYRLEEALPAEYKDTLDALYLEGVEGLDQSFLKNNTVNIQILC SI FNHDDPNKIAEEYYGFLMTKEYKNMGFSIKKLRECMLELPELSGYKEDQYNSVRSKLYKLFDFI IAHYFRKHPEKGEEMVDC LRLCMTEDEKDSHYEGTAKKLVRELAYDMQEAAEQANGSNITQMQKNEQQGKTKGMFAIRDEIRVSRKPVSYFSKVIYVMTLLL DGKEINDLLTTLINKFENIVSFEDVLRQLNVDCTFKPEFAFFGYDRCRNI SGELRLINSFARMQKPSAKAKHVMYRDALRILGL DNGMSEEALDQEVRRILQIGADGKPIKNANKGFRNFIASNVIESSRFRYLVRYNNPHKTRMIAQNEAIVRFVLSEI PDEQIRRY YDVCRDPKLPRSSSREAQVDILTGIITDWYRIFEDVPQSKKINKDRPDANDRMTLKKQRYQAIVSLYLTVMYLVTKNLVYVNS RYVMAFHALERDAYLYGITNIKGDYRKLTDNLLADENYKKFGHFKNKKWRGIAEQNLRNSDVPVIKSFRNMAAHISVIRNIDLY IGDIQKVDSYFALYHFLMQKLIQRWPENTKGLSDQTKKYYDALEQYNTYCKDFVKAYCTPFAYVTPRYKNLTIDGLFDRNRPG EDK (SEQ ID NO: 21)
>3300018493 I GaOl 87909_10024847_5
[mammals-digestive system- feces]
MGVEKNKVFESVIMNFDQERKYGFIEYKETNNLFFHMENVKNPKEIVKGAKVRFEIYENPKPKKQNQRFSAINVEVITDETHKE AKIQKNEFKTFDQFTKELQETQKVNGETKKEHITKNKHTNVKAAGVKSVFAVDDGNVLITSFGRGNAADIETLKSDDDKTINLT ETENQKKYVVTNKRSNVKGLADNPTKVESI IPGETQIGFKSILEKHFFGRTFNDNIHIQI IHNILDIKKILAVHTNNIVYALDN IHERGRENSAEKPIDMIGAGGI STSKEYEQYCSEKSDYEDNFLKQLINNERIAYFGNAFFKDEGNKKVMRTEKEIYYILGMLNE VRNVSTHFTEEDNRDWAKANLYNLSNRLKVGSKEVLNQLYKEKIDKIDANGFWKGCKRDFSILFKSLNLTTDKDKGELWGFY DFSIRKNYKNIGFSIKTLREYMLKISNSTLCADTISNNAIRPKAYKLYDFIIWHYYMNKPDKINDFVEKLRTQNKNDEKIKLYY DEAVCLLSELGREIHTMTSCVHNIENTSYEITDKKQKEYYKMQINSLNSADKVSDFSKVIYLVTLFLDGKEINDLLTTLINKFD NIASLLSVLEKQSGKKVEFVENYSFFNSSNLLKEKTLNKSENYTCKIVEELREINSFARMTGDCKIRKSAFEDASQLLGYHDKT WNLFEVLRLKELESKDWKKRTDDEQQEYDRLLNKHHYFKSGKKLPDTGLRNFI INNVIESRRFNYIVRYADPKKIRKCTENNE LLKFAFKDVPDSQVDRYYNICVTNKITNATREEKIERLVDIIKSMNLSKVATVKQRDKQDNVEKQKQLAIMSLYLNILYQIAKN LVYVNSRYVMAFHSLERDSQMLFDAYYDVKRGYCDLSTVLLFGVDDLQNRNRGSYKYLRDNRRSNKDVIETFGDFKGKVSKWE KKNQGLTNEIYDSLCNVAGTTKTEVQNEIKSILKSNGLDESASSYLSHKLWKVHSYKYLKQNLDCADNTMINQFRNNVAHLNT IRNMDGIENVTGITSYFQIYHYLMQKALYKEFKKCRENAVRKWI PYITENAEPKYVYWNKKEQQEVEVSFNPKI FGYMENIKNH SNTYCKDFVKALCAPFAYNLPRFKNLSIEELFDMHELSEEPKESMKLTD (SEQ ID NO: 22)
>WP_074833651.1
[ Ruminococcus albus]
MAKKSKGMSLREKRELEKQKRIQKAAWSVNDTPEKTEEANWSWVRTSAENKHSKKSAAKALGLKSGLVIGDELYLTSFGRG NEAKLEKKISGDTVEKLGIGAFEVAERDESTLTLESGRIKDKTARPKDPRHITVDTQGKFKEDMLGIRSVLEKKIFGKTFDDNI HVQLAYNILDVEKIMAQYVSDIVYMLHNTDKTERNDNLMGYMSIRNTYKTFCDTSNLPDDTKQKVENQKREFDKIIKSGRLGYF GEAFMVNSGNSTKLRPEKEIYHIFALMASLRQSYFHGYVKDTDYQGTTWAYTLEDKLKGPSHEFRETIDKIFDEGFSKISKDFG KMNKWLQILEQMIGELYGSIERQNLTCDYYDFIQLKKHKYLGFSIKRLRETMLETTPAECYKAECYNSERQKLYKLIDFLIYD LYYNRKPARIEEIVDKLRESWDEEKESIYSVEAKYVYESLSKVLDKSLKNSVSGETIKDLQKRYDDETANRIWDISQHSISGN WCFCKLIYIMTLMLDGKEINDLLTTLVNKFDNIASFIDVMDELGLEHSFTDNYKMFADSKAICLDLQFINSFARMSKIDDEKS KRQLFRDALVILDIGNKDETWINNYLDSDIFKLDKEGNKLKGARHDFRNFIANNVIKSSRFKYLVKYSSADGMIKLKTNEKLIG FVLDKLPETQIDRYYESCGLDNAVVDKKVRIEKLSGLIRDMKFDDFSGVKTSNKAGDNDKQDKAKYQAII SLYLMVLYQIVKNM IYWSRYVIAFHCLERDFGMYGKDFGKYYQGCRKLTDHFIEEKYMKEGKLGCNKKVGRYLKNNI SCCTDGLINTYRNQVDHFAV VRKIGNYAAYIKSIGSWFELYHYVIQRIVFDEYRFALNNTESNYKNSI IKHHTYCKDMVKALNTPFGYDLPRYKNLSIGDLFDR NNYLNKTKESIDANSSIDSQ (SEQ ID NO: 23)
>WP_041337480.1
[Ruminococcus bicirculans]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIA
AGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEIIPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDICT
VDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 24)
>DBYI01000091_43
[Ruminococcus flavefaciens ]
MKKKIKARDLREAKKQEKLAAFSAKANTVYENEDKNVEAFPEALNLRSIKKSMNKAAGLKSTLIDGKSLYLTAFGKGNNAWEH MIATDDSYSLKTLENEPSLKVKAADELKVTFMSRRPFVQESELSAVNPLHSGKDKPNKSAGQDMLGLKSELEKRYFGKIFDDNL HIQI IHNILDIEKI IAVYATNITAAIDHMVDDDNEQYLQGDFIGYMNTLNTYEVFMEPSKNPRLDSNARKNIENSREKFEYLLD TQRLGYLSLEYDKRSKDKRKSEEIKKRLYHLVAFAGQLRQWSFHSVEGLPRTWIYQLDNPKLAQEYRDTLDYFFNERFDAINKD FIETNNINLHILKEVFPAEDFQKLAALYYDFIVKKTFKNIGFSIKNLREQMLECDEAEKIRSKDMNSVRSKLYKLFDFCI FYQY FIDEERSRENWYLRSTLNDEQKDAFYEEEGKRLWSENRKKFIYFCDNINKWVKNDYSDEVAKCIDLNEFRVNSNVSYFSKLLY AMSFFLDGKEINDLLTTLINKFDNIRSFIDTANFLNIDVKFTKDYDFFNI ICDYAGELNI IKNIARMKKPSPSAKKNMYRDALT ILGIPTEMSDEQLDAEIDKILEKKINPVTGKTEKGKNPFRNFIANNVIENKRFIYVIKFCNPKNVRKLWNTKVTEFVLKRMPE TQIDRYFESCIEGNLNPTTEKKIEKLAEMIKNIKFEEFRNVKQKVRDNSQEAVEKERFKAIIGLYLTVIYLLVKNLWWSRYV MAFHCLERDAKLYGVQNIGGDYLALTAKLCAEGDDYGKKLSEAKQNINQDKVQMPKNYFLARNKRWREAIEQDIDNAKKWFIGE KFNNVKNYRNNVAHLTAIRNCAEFIGEITKIDSYFALYHYLIQRQLAGRLDPNHPGFEKNYPQYAPLFKWNTYVKDMVKALNSP FGYNIPRFKDLSIDALFDRNEMKEETDDEKKIQT (SEQ ID NO: 25)
>WP_075424065.1
[ Ruminococcus flavefaciens ]
MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSWEGEAFSAEMADKNAGYKIGNAKFSHPKGYAWAN NPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDI IGFGKFSTVYTYDE FKDPEHHPAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDILALLSGLRHWWHNNEEESRI SRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANWYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVM LDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSFNDDQKDALYYDEAN RIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPL IGWAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDENGNKLKKGKHGM RNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAWKFVLGRIADIQKKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKI ITGMNYDQFDKKRSVIEDTGRENAEREKFKKI ISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKG FSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTRQINREKAKTALNAYLRNTKWNV IIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDIAEVNSYFQLYHYIMQRI IMNERYEKSSGKVSEYFDAVNDEKKYNDRL LKLLCVPFGYCI PRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 26)
>WP_009985792.1
[Ruminococcus flavefaciens FD-1]
MKKKMSLREKREAEKQAKKAAYSAASKNTDSKPAEKKAETPKPAEI ISDNSRNKTAVKAAGLKSTI ISGDKLYMTSFGKGNAAV IEQKIDINDYSFSAMKDTPSLEVDKAESKEISFSSHHPFVKNDKLTTYNPLYGGKDNPEKPVGRDMLGLKDKLEERYFGCTFND NLHIQI IYNILDIEKILAVHSANITTALDHMVDEDDEKYLNSDYIGYMNTINTYDVFMDPSKNSSLSPKDRKNIDNSRAKFEKL LSTKRLGYFGFDYDANGKDKKKNEEIKKRLYHLTAFAGQLRQWSFHSAGNYPRTWLYKLDSLDKEYLDTLDHYFDKRFNDINDD FVTKNATNLYILKEVFPEANFKDIADLYYDFIVIKSHKNMGFSIKKLREKMLECDGADRIKEQDMDSVRSKLYKLIDFCI FKYY HEFPELSEKNVDILPAAVSDTKKDNLYSDEAARLWSIFKEKFLGFCDKIWWVTGEHEKDITSVIDKDAYRNRSNVSYFSKLMY AMCFFLDGKEINDLLTTLINKFDNIANQIKTAKELGINTAFVKNYDFFNHSEKYVDELNIVKNIARMKKPSSNAKKAMYHDALT ILGI PEDMDEKALDEELDLILEKKTDPVTGKPLKGKNPLRNFIANNVIENSRFIYLIKFCNPENVRKIWNTKVTEFVLKRI PD AQIERYYKSCTDSEMNPPTEKKITELAGKLKDMNFGNFRNVRQSAKENMEKERFKAVIGLYLTWYRWKNLVDWSRYIMAFH SLERDSQLYNVSVDNDYLALTDTLVKEGDNSRSRYLAGNKRLRDCVKQDIDNAKKWFVSDKYNSITKYRNNVAHLTAVRNCAEF IGDITKIDSYFALYHYLIQRQLAKGLDHERSGFDRNYPQYAPLFKWHTYVKDWKALNAPFGYNIPRFKNLSIDALFDRNEIKK NDGEKKSDD (SEQ ID NO: 27)
>CDC65743.1
[Ruminococcus sp . CAG:57]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKDEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRNESSNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRGDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 28)
>DJXD01000002_3
[Ruminococcus sp . UBA7013]
MKKQKSKKTVSKTSGLKEALSVQGTVIMTSFGKGNMANLSYKIPSSQKPQNLNSSAGLKNVEVSGKKIKFQGRHPKIATTDNPL FKPQPGMDLLCLKDKLEMHYFGKTFDDNIHIQLIYQILDIEKILAVHVNNIVFTLDNVLHPQKEELTEDFIGAGGWRINLDYQT LRGQTNKYDRFKNYIKRKELLYFGEAFYHENERRYEEDIFAILTLLSALRQFCFHSDLSSDESDHVNSF LYQLEDQLSDEFKE TLSILWEEVTERIDSEFLKTNTWLHILCHVFPKESKETIVRAYYEFLIKKSFKNMGFSIKKLREIMLEQSDLKSFKEDKYNSV RAKLYKLFDFIITYYYDHHAFEKEALVSSLRSSLTEENKEEIYIKTARTLASALGADFKKAAADWARNIRDYQKKANDYRI SF EDIKIGNTGIGYFSELIYMLTLLLDGKEINDLLTTLINKFDNII SFIDILKKLNLEFKFKPEYADFFNMTNCRYTLEELRVINS IARMQKPSADARKIMYRDALRILGMDNRPDEEIDRELERTMPVGADGKFIKGKQGFRNFIASNVIESSRFHYLVRYNNPHKTRT LVKNPNWKFVLEGIPETQIKRYFDVCKGQEIPPTSDKSAQIDVLARIISSVDYKIFEDVPQSAKINKDDPSRNFSDALKKQRY QAIVSLYLTVMYLITKNLVYWSRYVIAFHCLERDAFLHGVTLPKMNKKIVYSQLTTHLLTDKNYTTYGHLKNQKGHRKWYVLV KNNLQNSDITAVSSFRNIVAHI SWRNSNEYI SGIGELHSYFELYHYLVQSMIAKNNWYDTSHQPKTAEYLNNLKKHHTYCKDF VKAYCI PFGYWPRYKNLTINELFDRNNPNPEPKEEV (SEQ ID NO: 29) >SCH71549.1
[gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRNESSNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRGDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 30)
>SCJ27598.1
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKDNSNIQLGGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIED NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDI AAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISGILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIC TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 31)
>NZ_ACOK01000100_5
[ Ruminococcus flavefaciens FD-1]
MSAMTKGLRNCKGCWMKKKMSLREKREAEKQAKKAAYSAASKNTDSKPAEKKAETPKPAEI ISDNSRNKTAVKAAGLKSTI IS GDKLYMTSFGKGNAAVIEQKIDINDYSFSAMKDTPSLEVDKAESKEISFSSHHPFVKNDKLTTYNPLYGGKDNPEKPVGRDMLG LKDKLEERYFGCTFNDNLHIQI IYNILDIEKILAVHSANITTALDHMVDEDDEKYLNSDYIGYMNTINTYDVFMDPSKNSSLSP KDRKNIDNSRAKFEKLLSTKRLGYFGFDYDANGKDKKKNEEIKKRLYHLTAFAGQLRQWSFHSAGNYPRTWLYKLDSLDKEYLD TLDHYFDKRFNDINDDFVTKNATNLYILKEVFPEANFKDIADLYYDFIVIKSHKNMGFSIKKLREKMLECDGADRIKEQDMDSV RSKLYKLIDFCI FKYYHEFPELSEKNVDILRAAVSDTKKDNLYSDEAARLWSIFKEKFLGFCDKIVVWVTGEHEKDITSVIDKD AYRNRSNVSYFSKLMYAMCFFLDGKEINDLLTTLINKFDNIANQIKTAKELGINTAFVKNYDFFNHSEKYVDELNIVKNIARMK KPSSNAKKAMYHDALTILGI PEDMDEKALDEELDLILEKKTDPVTGKPLKGKNPLRNFIANNVIENSRFIYLIKFCNPENVRKI WNTKVTEFVLKRI PDAQIERYYKSCTDSEMNPPTEKKITELAGKLKDMNFGNFRNVRQSAKENMEKERFKAVIGLYLTWYRV VKNLVDWSRYIMAFHSLERDSQLYNVSVDNDYLALTDTLVKEGDNSRSRYLAGNKRLRDCVKQDIDNAKKWFVSDKYNSITKY RNNVAHLTAVRNCAEFIGDITKIDSYFALYHYLIQRQLAKGLDHERSGFDRNYPQYAPLFKWHTYVKDWKALNAPFGYNIPRF KNLSIDALFDRNEIKKNDGEKKSDD (SEQ ID NO: 200)
>3300006226 I Ga0099364_10024192_5
[ arthropoda-digestive system-cubitermes and nasutitermes termite gut]
MGVKSVLAHGKDEKGHIKLAITAFGKGNKAELAIQTDEKGSNLAKTYKERNITANKIVSEGIQTSGTIAGEGHATFLNNPAEHV GTDYLKLKETLEMEFFGKSFPGDSVRIQIIHQILDIQKLLGIYITDIIYCINNLRDETHLDHESDIVGLSMSNTNVNLALNQMR PYFGFFGEAFRPVGDDKVKEITLSDEVRKNIEKI IALEEQKRNPSTPRFKQENINLEIENAMGKFKSKDAFETAKKKYNRIVAD ETNAKTLRILGAMRQITAHFKDQATLFMSDVELPKILKKEFSKADWQTVEDYYAKLVDRINEGFCKNAATNVHFLTELLPEESK KQLTEDYFRFAILKEGKNLGWMKRLREVMFALFVPELTAPETKKRYDSYRAKIYGLTDFLLFKHIHNTKQLEEWVAVLRETSN EDAKENLYDEFARTAWNTVGDSAKQLIENMQSYFTKKEKEITKTAQPVLSTSSIAHTSKKITQFSSFAKLLAFLCNF EGKEIN ELLSAYIHKFENIQEFINLLEKLEGKKPQFTENYALFNEAAGQRAGEIAQNLRILASIGKMKPDLGDAKRQLYKAAIEMLGIDT EEYI SDEWLEPNMLLAQPPKEPKKDNEKYRKEPHKYSYEKDMETYRKKLREYEETWRSLIDYEYLMPETNPFRNFVAKQVIESR RFMYLVRYTKPKTVPALMSNRAIVHYVLSRIADIQDHHMTESQIDRYYQNLPQYNEQQHKNVSLETKIDALADYLCKYTFEKNV LKQKNGIVLNTKSATKNVEIEHLKALTGLYLTVAYIAVKNLVKANARYYIAFSI FERDYALFEKKLGKDTLEKYVKPFKYIDKG EEKEGKNNFFALTEYLLDKDNSLRYQWNNDLSDEENKQALRKHLDKKEIRSQRHFSQYWLDI FARQIENAKKTSESGYLLTAAR NCALHLNVLTALPEFVGEFRKTGDKMTSYFELYHFLLQKLMLAEAGLNLDEYRERIDTYQTACKDLINITYVSLGYNLPRYKNL TCEPLFDEESATGKERQTRLDEKSKEKKQRKGGQK (SEQ ID NO: 201) >CDZK01015063_14
[gut metagenorae]
MAKKNKMKPRERREAQKKARQLKAAEINNNAVPAIAAMHAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNQTQLSSKDNSNIELCGVTKWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIYPEYRDTLDYLVEERLKSINKDFIQGN
KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDW
AGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKKYIGD
IRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 202)
>CEAA01017658_2
[gut metagenorae]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 203)
>OCTW011587266_5
[gut metagenorae]
MKQNDRENNNKIKKSAAKAVGVKSLARLSDGSTWSSFGKGAAAELESLITGGEIRKLSDKAILEITDDTQNKNAYNVKSSRIP NLTARTDKLSDKSGMDDLGFKRELELEVFGQCFDDSIHIQIAHAVFDIQKSLAAVI PNVLYTLNNLDRSYSTDNTSDKKDIIGN TLNYQHSYESFNVEKRGEFTEYYNAAKDRFSYFPDILCVLEKWGKDRYQPKSEKDAFNVLSSVNMLRNSLFHFAPKSNDGKAR IAVFKNQFDSDFSHITSTWKIYSAKIAGVNENFLNNEGNNLYI ILKATNWDIKKIVPQLYRFSVLKSDKNMGFNMRKLREFAV ESKNIDLSRLNDKFLTNNRKKLYKVIDFIIYYHLNKVLKDSFVDDFVAALRASQSEEEKEKLYAQYSERLFADEGLKSAIKKAV DMISDTKSNIFKMKTPLDKALIENIKWSDASDFCKLIYVFTRFLDGKEINILLNSLIKKFQDIHSFNTTVKKLSENNLIINAD YVDDYSLFEQSGTVARELMLIKSI SKMDFGLDNINLSFMYDDALRTLGVSDENLPEVKREYFGKTKNLSAYIRNNVLENRRFKY VIKYIHPSDVQKIACNKAIAGFVLNRMPDTQIKRYYDSLINKGATDIQAQAKALLDCITGISFDAIKDDKHLHKSKEKSPQRSA DRERKKAMLTLYYTIVYI FVKQMLHINSLYTIGFFYLERDQRFIYSRAKKENKNPSKNSYLNDFRSVTAYFI PSEIMKRIEKNE NKGFLEDFEALWNSCGKTSRLRKEDVLLYARYISPDHALKNYKMILNSYRNKIAHINVIMSAGKYTGGIKRMDSYFSVFQHLVQ CDILSNPNNKGKCFESESLKPLLLDMKFDGTDEKLYSKRLTRALNI PFGYNVPRYKNLTFEKIYLKSSINE (SEQ ID NO: 204)
>OCW011003687_3
[gut metagenorae]
MAKKSKGMKPKEKRELEKQKRIQKAWKSADDTPVKAEATKAVSWTDLSVENKHNKKSAAKALGLKSGLVIGDDLYLTSFGRG NEAKLEKKISGETVENLGIGAFEVTERDESTLTLESGRIKDKTARPKDPRHITVDAQGKFKEDMLGIRSVLEEKIFVGKKFNDN IHVQLAYNILDIEKIMAQYVSDIVYMLHNTDKTERNDNLMGYMSIQNPYSVFCNPNFSAAKTQRNWRQKQELDNIIKSGRLGY FGEAFMVYSGNSSKLRPEKEIYHI FALMASLRQSYFHGYVKDTDYQGPTWPYTLEDKLKDPSHEFRETLDKI FDEGFSKI SKNF GKQNNVNLQILEEMLGELYGSTDSKSLACDYYDFIQLKKHKYLGFSIKRLRETMLETTPAACYKAECYNSIRHKLYLLIDFLIY DLYYNRKPARIEEIVDKLRESVNDEEKESIYSAETKYVYEALGKVLVRSLKKYLNGATIRDLKNRYDAKTANRIWDISEHSKSG HVNCFCKLIYMMTLMLDGKEINDLLTTLVNKFDNIASFIDVMDELGLEHSFTDNYKMFADSKAICLDLQFINSFARMSKIDDEK SKRQLFRDALWLDIGDKNEDWIEKYLTSDIFKRDENGNKIDGEKRDFRNFIANNVIKSARFKYLVKYSSADGMIKLKKNEKLI SFVLEQLPETQIDRYYESCGLDCAVADRKVRIEKLTGLIRDMRFDNFRGVNYSNDACKKDKQAKAKYQAI ISLYLMVLYQIVKN MIYVNSRYVIAFHCLERDLLFFNIELDNSYQYSNCNELTDMFIKDKYMKEGALGFNMKAGRYLTKNIGNCSNELRKIYRNQVDH FAWRKIGNYAADIKSIGSWFELYHYVMQRIVFDEYRFALNNTESNYKNS11KHHTYCKDMVKALNTPFGYDLPRYQNLSIGDL FDRNNYLNKTKESIETKSPIDNP (SEQ ID NO: 205)
>OCW011003687_3
[gut metagenorae]
MVQREGCVMAKKSKGMKPKEKRELEKQKRIQKAWKSADDTPVKAEATKAVSWTDLSVENKHNKKSAAKALGLKSGLVIGDDL YLTSFGRGNEAKLEKKISGETVENLGIGAFEVTERDESTLTLESGRIKDKTARPKDPRHITVDAQGKFKEDMLGIRSVLEEKIF VGKKFNDNIHVQLAYNILDIEKIMAQYVSDIVYMLHNTDKTERNDNLMGYMSIQNPYSVFCNPNFSAAKTQRNWRQKQELDNI IKSGRLGYFGEAFMVYSGNSSKLRPEKEIYHI FALMASLRQSYFHGYVKDTDYQGPTWPYTLEDKLKDPSHEFRETLDKI FDEG FSKI SKNFGKQNNVNLQILEEMLGELYGSTDSKSLACDYYDFIQLKKHKYLGFSIKRLRETMLETTPAACYKAECYNSIRHKLY LLIDFLIYDLYYNRKPARIEEIVDKLRESVNDEEKESIYSAETKYVYEALGKVLVRSLKKYLNGATIRDLKNRYDAKTANRIWD ISEHSKSGHVNCFCKLIYMMTLMLDGKEINDLLTTLWKFDNIASFIDVMDELGLEHSFTDNYKMFADSKAICLDLQFINSFAR MSKIDDEKSKRQLFRDALWLDIGDKNEDWIEKYLTSDIFKRDENGNKIDGEKRDFRNFIANNVIKSARFKYLVKYSSADGMIK LKKNEKLI SFVLEQLPETQIDRYYESCGLDCAVADRKVRIEKLTGLIRDMRFDNFRGVNYSNDACKKDKQAKAKYQAI ISLYLM VLYQIVKNMIYVNSRYVIAFHCLERDLLFFNIELDNSYQYSNCNELTDMFIKDKYMKEGALGFNMKAGRYLTKNIGNCSNELRK IYRNQVDHFAWRKIGNYAADIKSIGSWFELYHYVMQRIVFDEYRFALNNTESNYKNSIIKHHTYCKDMVKALNTPFGYDLPRY QNLSIGDLFDRNNYLNKTKESIETKSPIDNP (SEQ ID NO: 206)
>ODAI010069496_4
[gut metagenorae]
MAKKIKPRDLRESKRQEKLAAYSVKANEKKTVHTTEEKPAAVLTVTASENKKNKKTSNKAAGLKSTLVYGNKLYITSFGKGNEA IIEQKVDTSDYSFSDVRSDPSLKIKSADDVSI SFSSERPFINKSLLTAWPLHSGKDKPKRAAGQDMLGLKSELEKRYFGKTFD DNIHIQLIHNILDIEKIFAVYSANIVAALDHMIDGDDKEYLENDFIGYMNTLNTYEVFMDPSKVFSDCDNRKKNIDKSREKFET LIDSKRLRYFGFEYDPDGKNKNEEMKKRLYHLVAFAGQLRQWSFHSEGNFQLEWLYKLDDSRIAQEYRDTLDYFFDRRFDELNN NFVEQNATNLFILKETFPGEDLKAVTDLYYDFIIVKSQKNIGFSIKKLREKMLGTEEAAPIKAHDMDSYRPKLYKLIDFCIFKH YHEYTEISEKNVDTLRAAVSEEQKESFYADEAKRLWGI FDKQFLGFCKKINVWVNGSHEKEILGYIDKDAYRRKSDVSYFSKFL YAMSFFLDGKEINDLLTTLINKFDNIASFI STAKELDAEIDRILEKKLDPVTGKPLKGKNSFRNFIANNVIENKRFIYVIKFCN PKNVLKLVKNTKVTEFVLKRMPESQIDRYYSSCIDTEKNPSVDKKI SDLAEMIKKIAFDDFRNVRQKTRTREESLEKERFKAVI GLYLTWYLLIKNLWWSRYVMAFHCLERDAKLYGINIGKNYIELTEDLCRENENSRSAYLARNKRLRDCVKQNIDNAKNMKS KEKQRVFFKDYSTWPFLEKVFYYGSFSSADFEEMDMMKKSKYSYYKRILEYAFGDLLFERKNI SKTN (SEQ ID NO: 207)
>ODAI011611274_2
[gut metagenorae]
MKKKISLKEQRNTKKAENKLKYQKAQAERAAAAQQTAAGAESEENPCFDWKDTKRKALNPLHVEIEAPSAKKSSVKANGLKSL LLTDGKTVMTSFGRGSEANVEKRFDETGTKTFDRDPELFSAKPLETGYRIQRFNASPKDAGLAYRPAGVRPDQIGAKAALEKRY FGKETPGDNIHVQIAYQIQDIEKLLAVYISNI IYAVNNVTGVSAMKDSKGRPVDLLGDYGILGEEGLTKRLQRI PEQADEEAKA LQAFLCSERLSYFGKEFCLVRNSPKQPDKEEKRQYKLMRVLCLLGELRQFLVHGKKKEKEFAWLYRLDRQLSQEYRKLLGEFYD AQVDKVNKSFLTNSTVNLEVLFRALKTGTDPERKTVTQEYYQFTVRKEDGNLGFSLKTLREILLSAYKHEVRDKEYDSIRHKLY QLFSFALYHYYKTGVGAERREAFVAKLRAVMTAEAKQRAYADEAAEIWNDEGSGIRAAFLEILEAVDFGSAVKGIKARSSVAGD KRFAEWLEEVRIRPEGVSCFTKLMYLLTRFLDGKEINELLTGLINKLENIQSFLDVMQQEHAETGLSDAFSFFEYSGEIAAELR MTRSFARMAAADPEAKRFMWDGAKLLGFNPKDTESEDEGIIPAIYGDACAEYLQFSEEEKEAFYVQEGLYGKEREKFSPYAYF HTDTSLRNFIAKNWESARFRYVIRYVSPEIARKYARQEALVRFALHRVPLLQLRRYYQSCCGPKKDPDAAECVDFLAGVWRV DFANFTDVRTGDSSKSEQEKKQKYQAIVGLYLTWYWIVKNLWWSRYVMAFHILERDTVLLEGKRLFVGGMKAEDPFLLTDG YVSRQDAYVRKRIGENKRANRHGLNCVLENRNALGSDPASTDAAASLIWSYRNAAAHLTAVAAAQEYVSELREIHSYFEVYHYA MQRYLKSGAEFAELVSKNGPASGKIAAWANAVDRCHSFCKDWLWLLNVPFAYNPARYKNLSIANLFDKNEAAPVTEDASEQKED
E (SEQ ID NO: 208)
>OATA01000148_47
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIA
AGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQSKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEIIPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDICT
VDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 209)
>OAVJ01001264_7
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLEYEVDK VDNNVYNQTQLSSKGSSNIKLCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNIHI QLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLLKTKRL GYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQGNK WISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDVI AGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAMLTMFRDALT ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC VEVPDMNSSLEAKRSELARMIKSISFDDFKNVKQQAKGRENVAKEMAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG LYKEII SELASKNLKNDYRILSQTLCELCDNCDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGD IRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 210)
>OBAE01000973_3
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAVEINNNAVPEIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKNSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND VIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIYAVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDNLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 211)
>OBAR01000289_55
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSIFVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKGSSNIELHGVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDPEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEVPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 212)
>OBCV01000332_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 213)
>OBDE01000870_1
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKGSSNIELHGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIEG NKINISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNHYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKVVMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLVNFVMIVMSRRICS (SEQ ID NO: 214)
>OBII01002626_5
[human gut metagenorae]
MKSILVSKNKMYITSFGKGNSAVLEYEVDNNDYNKTQLSSKDNSNIELRGVTKVNITFSSKHGLESGVEINTSNPTHRSGESSP VRWDMLGLKSELEKRFFGKTFDDNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDN SSLSDDKKANVRKSLSKFNALLKTKRLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNID PEYRETLDYLVDERFDSINKDFIQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQ YDSVRSKMYKLMDFLLFCNYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMD FDEKILDSEKKNASDILYFSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNEL FIVKNIASMRKPAASAKLTMFRDALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREV AKNEKWMFVLGGI PDTQIERYYKSCVEFPDMNSSLEVKRSELARMIKNI SFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVM YLLVKNLVNVNARYVIAIHCLERDFGLYKEII PELASKNLKNDYRILSQTLCDDRDESPNLFLKKNKRLRKCVEVDINNADSSM TRKYRNCIAHLTWRELKEYIGDIRTVDTYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIP RFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 215)
>OBII01002626_3
[human gut metagenome]
MYITSFGKGNSAVLEYEVDNNDYNKTQLSSKDNSNIELRGVTKVNITFSSKHGLESGVEINTSNPTHRSGESSPVRWDMLGLKS ELEKRFFGKTFDDNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKAN VRKSLSKFNALLKTKRLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYL VDERFDSINKDFIQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYK LMDFLLFCNYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEK KNASDILYFSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMR KPAASAKLTMFRDALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFV LGGI PDTQIERYYKSCVEFPDMNSSLEVKRSELARMIKNI SFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNV NARYVIAIHCLERDFGLYKEII PELASKNLKNDYRILSQTLCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAH LTWRELKEYIGDIRTVDTYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQL FDRNEYLTEK (SEQ ID NO: 216)
>OBJF01000033_8
[human gut metagenome]
MAKKKRITAKERKQNHRELLMKKADSNAEKEKAKKPWENKPDTAI SKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFGLNYRVPYSEYGGGKDSNGEPKNKFKWEKRDNFSKFYNESK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKILLEIVEALRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFISHNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAS QEPLLNDALLSLGVSDDTKVLENTYKKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPGCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNDVDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREKYNKFFTAYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFNYCKEKNDWLAKMKDLAHIKSDCNEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQWLKHYENLNDFIRILY (SEQ ID NO: 217)
>OBJF01000033_8
[human gut metagenome]
MAKKKRITAKERKQNHRELLMKKADSNAEKEKAKKPWENKPDTAI SKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFGLNYRVPYSEYGGGKDSNGEPKNKFKWEKRDNFSKFYNESK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKILLEIVEALRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFISHNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAS QEPLLNDALLSLGVSDDTKVLENTYKKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPGCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNDVDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREKYNKFFTAYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFNYCKEKNDWLAKMKDLAHIKSDCNEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQ (SEQ ID NO: 218) >OBKG01000025_26
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVDEVNITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 219)
>OBKR01000858_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKYNSNIELGDVNEVNITFSSKHGFGSGMKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 220)
>OBVH01003037_1
[human gut metagenome]
MAKKKRITAKERKQNHRELLMKKADSNAEKEKAKKPWENKPDTAI SKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFGLNYRVPYSEYGGGKDSNGEPKNQSKWEKRDNFIKFYNESK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKLLLEIVETLRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFARKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISNNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP QEPLLKDALLSLGVSDDTKVLENTYNKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPGCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNDVDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREKYNKFFTAYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFNYCKEKNDWLAKMKDLAHIKSDCNEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQWLKHYENLNDFIRILY (SEQ ID NO: 221)
>OBVH01003037_2
[human gut metagenome]
MAKKKRITAKERKQNHRELLMKKADSNAEKEKAKKPWENKPDTAI SKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFGLNYRVPYSEYGGGKDSNGEPKNQSKWEKRDNFIKFYNESK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKLLLEIVETLRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFARKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISNNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP QEPLLKDALLSLGVSDDTKVLENTYNKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPGCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNDVDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREKYNKFFTAYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFNYCKEKNDWLAKMKDLAHIKSDCNEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQ (SEQ ID NO: 222)
>OBVY01000267_8
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEVNITFSSKHGFGSGMKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEI IKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 223)
>OBXZ01000094_20
[human gut metagenorae]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNIPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRVPYSEYGGGKDSNGEPKNKSKWEKRKNFIKFYNKSK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKNDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSIKHKLYKTYDFVITHY LNSNDKLLLEIVEALRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFAEKGNKLFKEMVIIKKEYVEDI SIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISNNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIVRMENKIENAP QEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKADKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSN IPEEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNGVDYLLLPEIFSGSKYREQTKNLKLPKEKDRDIMKKYLPNDKDREGYNKFFRAYRNNIVHL NIIAKLSELTSNIDKDINSYFDIYHYCTQRVMFNYCKENNNIVLAKMKDLAHIKSDCDEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQWLNH (SEQ ID NO: 224)
>OBXZ01000094_20
[human gut metagenome]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNIPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRVPYSEYGGGKDSNGEPKNKSKWEKRKNFIKFYNKSK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKNDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSIKHKLYKTYDFVITHY LNSNDKLLLEIVEALRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFAEKGNKLFKEMVIIKKEYVEDI SIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISNNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIVRMENKIENAP QEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKADKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSN IPEEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGYFYKDICGKKDKKKSYNGVDYLLLPEIFSGSKYREQTKNLKLPKEKDRDIMKKYLPNDKDREGYNKFFRAYRNNIVHL NIIAKLSELTSNIDKDINSYFDIYHYCTQRVMFNYCKENNNIVLAKMKDLAHIKSDCDEFSSKHTYPFSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQ (SEQ ID NO: 225)
>OCHB01002119_1
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 226)
>OCHC01000012_250
[human gut metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSEDSSNIELCGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 227)
>OCHN01000290_35
[human gut metagenorae]
MLCLKPTLEKKFFGKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSR EKADFDAFEKFIGNYRLAYFADAFYVDKNKSKSKPKDKAKGIQRGEKEIYSILALIAKLRHWCVHSEEGRAEF LYKLDELKSD FKNVLDWYNRPVEKINNRFIENNKVNIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSV RNKLYQMTDFILYTGYINEDSDRADDLVNTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVAASLDVKNINELKNNAFTIPDN ELRKCFISYADSVSEFTKLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEVMDELGLERTFTDEYSFFEGSTKYLAELVELNS FVKSCSFDINAKRTMYRDALDILGIESGKTEEDIEKMIDNIVQFDANGKKLPNKNHGLRNFIASNVIDSNRFEYLVRYGNPKKI RETAKCKPAVRFVLNEIPDAQIERYYKACYPDEKSLCFANMQRDKLAGVIANIKFDDFSDAGSYQKANATSTKITSEAEIKRKN QAIIRLYLTVMYIMLKNLWWARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGIIKTEFDKSLAENAAN RYFRNARWYKLILDNLKKSERAWNEFRNTVCHLNAIRNININIKEIKEVENYFALYHYLIQKHLEKRFADNGGSTGDFI SKLE EHKTYCKDFVKAYCTPFGYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 228)
>OCPQ01000020_138
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYITNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVIKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSPLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 229)
>OCPU01001206_17
[human gut metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSEDSSNIELCGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFCASALKSILIMQTAA (SEQ ID NO: 230)
>OFMU01000310_31
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKGSSNIELHGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIEG NKINISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNHYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 231)
>OFMV01000268_25
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAIDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 232)
>OGCM01002738_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSDGSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFVQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSPLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 233)
>OGCO01000353_15
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNVLL KTKRLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDIAAGEALVRKLRFSMTDDEKEGLYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 234)
>OGOK01000323_15
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN KVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIA AGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT ILGIDDKITDDRISEILKLKEKGKGIHGLRNFVTNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC VEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG LYKEII PELASKNLKNDYRILSQTLCELCDNPDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGD ICTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 235) >OGOL01000786_27
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEWITFSSRRGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEDDDESHDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKYSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 236)
>OGOO01001137_18
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAVEINNNAVPEIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKNSSNIELRGWEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLLKTK
RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG
NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND
VIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY
MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA
LTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYK
SCVEFPDMNSPLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD
FGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDI
RTVDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 237)
>OGOP01001824_10
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVDEWITFSSKHGFESGVKINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGLENESNNDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTKR LGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRETLDYLIDERFDSINKGFIQGN KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYHRKDW AGEALVRKLRFSMTDEEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTTGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT ILGIEDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKSC VEFPDMNSSMGAKRRELAKMIKSISFENFKDVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG LYKEII PELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEYIGD IYAVDSYFSIYHYVMQRCITKRENDTEQAEKIKYEDDLFKNHGYTRDFVKALNSPFGYNI PRFKNLSIKQMFDRNEYLTEK
(SEQ ID NO: 238)
>OGPB01000314_7
[human gut metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSEDSSNIELCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKKYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 239)
>OGPJ01000449_26
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSDGSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSPLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 240)
>OGPS01000624_23
[human gut metagenorae]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVENTVYMTSFGR GNDAVLEQKIVDTSHEQLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKARNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLNELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKRLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEDSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPENTALCSANKKREKLADMIAEIEFENFSDAGNYQKANVTSKTHEAEIKRKNQSI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSFAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 241)
>OGQH01000331_48
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKNSSNIELHGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSPLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 242)
>OGQO01007270_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKGSSNIELHGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRETLDYLIDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 243)
>OGQW01001429_6
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLEYEVDNND YNQTQLSSKGSSNIELHGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNIHIQLIYN ILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTKRLGYFGL EEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIEGNKINISL LIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNHYRNDVAAGEALV RKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYMLTYFLDG KEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALTILGIDD NITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKVVMFVLGGI PDTQIERYYKSCVEFPDM NSSLEAKRSEIA.RMIKNI SFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIRTVDS YFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 244)
>OGRA01000610_24
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELRDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDVAAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 245)
>OGRE01001635_6
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNVYNQTQLSSKGSSNIKLCGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYY RNDVIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAMLTMF RDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEVPDMNSSLEAKRSELARMIKSISFDDFKNVKQQAKGRENVAKEMAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCL ERDFGLYKEI ISELASKNLKNDYRILSQTLCELCDNCDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELK EYIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLT EK (SEQ ID NO: 246)
>OGRF01000967_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAVEINNNAVPEIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKNSSNIELRGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDNFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELNKYIK DIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 247)
>OGRN01001989_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDDNDYNQTQLSSKDNSNIELGDVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALL KTKRLGYFGLEEPKTKDNRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDF IEDNKVNI SLLIDMMKDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGESLVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLKAKRSELARMIKNISFEDFKDVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQMLCELCDDRDKSPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIYAVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDNLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 248)
>OGRQ01003333_5
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNQTQLSSKGSSNIELHGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNVLLKTK
RLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG
NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND
VAAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY
MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA
LTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK
SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD
FGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI
GDIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 249)
>OGRU01000829_2
[human gut metagenome]
MKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLEYEVDNN DYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGMKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNIHIQLIY NILDIEKILAVYVTNIVYALNNMLGEGDDSNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKKNIRKSLRKFNDLLKTKRLGYFGL EEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIYPEYRDTLDYLVDERFDSINKGFIQGNKVNISL LIDMMKGYEPDDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIAAGESLV RKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYMLTYFLDG KEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTVGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALTILGIDD KITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAENEKVVMFVLGGI PDAQIERYYKSCVEFPDM NSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELNKYIKDIRTVDS YFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 250)
>OGSD01001176_18
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSESSSNIELCGVTKWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDIYSFINNIDPEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFEYIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEVPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 251)
>OGWY01002732_3
[human gut metagenome]
MGKKIHARDLREQRKTDRTEKFADQNKKREAQRAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGRKKDEPEQSVPTDMLCLKPTLEKKFF GKEFDDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKARNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLNELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYCESIREVAEALDGDNIKRLSKSNIEIRDNELRKCFISYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLEKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIKFENFSDAGNYQKANVTSKTHEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADNGGSTGDYIGKLEEHKTYCKDFVKAYCTPFGY NLVRYKNLTIDGLFDKNYPGKDDSDEQK (SEQ ID NO: 252)
>OGXI01000433_6
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVDEVNITFSSKHGFESGVKINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGLENESNNDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTKR LGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRETLDYLIDERFDSINKGFIQGN KVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV IAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 253)
>OGXJ01002463_5
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKRGNESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYETDDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGTYADEAEKLWGKFRNDFENIADHMNGDAIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDIYNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 254)
>OGXL01002096_10
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDDNDYNKTQLSSKDNSNIELGNVNEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRDTLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKIMDFLLFCNYY RNDVVAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMF RDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCL ERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 255)
>OGYD01000683_23
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYITNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVIKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 256) >OGYL01002810_3
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASNRFILFSTM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAIDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 257)
>OGYY01000371_37
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNATPTIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKNSSNIELRGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFIGYLSARNTYKVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDPEYRETLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGEAIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNIGFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 258)
>OGZC01000639_10
[human gut metagenome]
MKKKNIRATREALKAQKIKKSQENEALKKQKLAEEAAQKRREELEKKNLAQWEETSAEGRRSRVKAVGVKSVFWGDDLYLATF GNGNETVLEKKITPDGKITTFPEEETFTAKLKFAQTEPTVATSIGI SNGRIVLPEI SVDNPLHTTMQKNTIKRSAGEDILQLKD VLENRYFDRSFNDDLHIRLIYNILDIEKILAEYTTNAVFAIDNVSGCSDDFLSNFSTRNQWDEFQNPEQHREHFGNKDNVICSV KKQQDLFFNFFKNNRIGYFGKAFFHAESERKIVKKTEKEVYHILTLIGSLRQWITHSTEGGI SRLWLYQLEDALSREYQETMNN CYNSTIYGLQKDFEKTNAPNLNFLAEILGKNASELAEPYFRFIITKEYKNLGFSIKTLREMLLDQPDLQEIRENHNVYDSIRSK LYKMIDFVLVYAYSNERKSKADALASNLRSAITEDAKKRIYQNEADQLWTSYQELFKRIRGFKGAQVKEYSSKNMPIPIQKQIQ NILKPAEQVTYFTKLMYLLTMFLDGKEINDLLTTLINKFDNI SSLLKTMEQLELQTTFKEDYTFFQQSSRLCKEITQLKSFARM GNPI SNLKEVMMVDAIQILGTEKSEQELQSMACFFFRDKNGKKLNTGEHGMRNFIGNNVI SNTRFQYLIRYGNPQKLHTLSQNE TWRFVLSRIAKNQRVQGMNGKNQIDRYYETCGGTNSWSVSEEEKINFLCKILTNMSYDQFQDVKQSGAEITAEEKRKKERYKA II SLYLTVLYQLIKNLVNINARYI IAFHCLERDAILYSSKFNTSINLKKRYTALTEMILGYETDEKARRKDTRTVYEKAEAAKN RHLKNVKWNCKTRENLENADKNAIVAFRNIVAHLWI IRDADRFITGMGAMKRYFDCYHYLLQRELGYILEKSNQGSEYTKKSLE KVQQYHSYCKDFLHMLCLPFAYCI PRYKNLSIAELFDRHEPEAEPKEEASSVNNSQFITT (SEQ ID NO: 259)
>OHAI01000724_7
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAVEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERYYKS CVEVPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTRDFVKALNSPFGYNIPRFKNLSIKQLFDRNEYLTEK
(SEQ ID NO: 260)
>OHAJ01000052_20
[human gut metagenome]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAI SKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRI IGDVSSDVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLTTHPNKPDKNSGMDALCLKIYFEKEIFKDKFNDNM HIQTIYNI FDIEKTLAKHITNI IYAVNSLDRSYIQSGNDTIGFGLNYRIPYAKYGRGKDSNGKPNNSNLKKRESFIKFYNNAKD RFGYFESVFYQNGKPI SREKLYIYLNILNFVRNSTFHYNNTSTYLYRKEYKYTDKDNCSVKEFEFVSYLNEFVKNKFKNVNKNF ISNEKNNLYI ILNAYGEDIEDVEVVKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSIKHKLYKT YDFVITHYLNSNDKLLLEIVEALRLSKNDDEKENVYKIYAEKIFKAEYVINPIKTI SNLFAEKGDKLFNEKVSI SEEYVEDIRI DKNIHNFTKVIFFLTCFLDGKEINDLLTNIISKLQVIEDHNNVIKAIANNNDAVYKDYSDKYAVFKNSGKIATKLEAIKSIARM ENKINKAFKEPLLKDAMLALGVSPNDLDEKYEKYFKTDVDADKDHQKVSTFLMNNVINNSRFKYWKYINPADINRLAKNKHLV KFVLDQIPHKQIDSYYNSVCTVEEPSYKGKIQLLTKKITGLNFYSLFENCKI PNVEKEKKKAVITLYFTI IYILVKNLVNINGL YTLALYFVERDGFFYKKICEKKDKKKTNKDVDYLLLPEIFSGSKYREETKNLKLPKEKDREIMKKYLPNDEDRKEYNKFFKQYR NNIVHLNI IANLSKLTSTIDKEINSYFEIFHYCAQRVMFDYCKNNNKVVLAKMKDLAHIKSDCDEFSSKYTYPYSSAVLRFMNL PFAYNVPRFKNLSYQKFFDKQRLEALEKNLNI (SEQ ID NO: 261)
>OHAN01001071_11
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNDYNQTQLSSKGSSNIELHGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDF IEGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCSYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRTLSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSIMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 262)
>OHAR01000226_9
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKVAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDKVDNDNYNKTQLSSKDNSNIELGDVDEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNVLL KTKRLGYFGLEEPKTKDTNALEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYY RNDVVAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMF RDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCL ERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELK EYIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLT EK (SEQ ID NO: 263)
>OHBL01000590_7
[human gut metagenome]
MAKKNKMKPRERREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAKNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNYYRNDV VTGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 264)
>OHBP01000023_129
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 265)
>OHBQ01000429_2
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNVYNQTQLSSKGSSNIKLCGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSIN KGFIQGNKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGYRFKDKQYDSVRSKMYKLMDFLLFCN YYRNDVVAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKI IDSEKKNASDLLYF SKMIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLT MFRDALTILGIDDNITDDRI SEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKVVMFVLGGIPDTQI ERYYKSCVEFHDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTWlYLLVKNLWWARYVIAIH CLERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRE LKEYIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEY LTEK (SEQ ID NO: 266)
>OHBW01001448_1
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNWEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 267)
>OHCE01000125_17
[human gut metagenome]
MAKKNKMKPRERREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDW
AGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDALT
ILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEIIPELASKNLKNDYRILSQTLCELCDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIRT
VDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 268)
>OHCH01000211_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSEDSSNIELCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFWKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 269)
>OHCP01000044_27
[human gut metagenorae]
MAKKITAKQKREEKERLNKQKWAKNDSVIIVPETKEEIKTGEIQDNNRKRSRQKSQAKAMGLKAVLSFDNKIAIASFVSSKNAK SSHIERITDKEGTTISWSKMFESSVNKRDINIEKRITIEEPQQDGTIKKEEKGVKSTTCNPYFKVGGKDYIGIKEIAEEHFFG RAFPNENLRVQIAYNI FDVQKILGTFWNI IYSFYNLSRDEVQSDNDVIGMLYSISDYDRQKETETFLQAKSLLKQTEAYYAYF DDVFKKNKKPDKNKEGDNSKQYQENLRHNFNILRVLSFLRQICMHAEVHVSDDEGCTRTQNYTDSLEALFNI SKAFGKKMPELK TLIDNIYSKGINAINDEFVKNGKNNLYILSKVYPNEKREVLLREYYNFVVCKEGSNIGISTRKLKETMIAQNMPSLKEENTYRN KLYTVMNFILVRELKNCATIREQMIKELRANMDEEEGRDRIYSKYAKEIYLYVKDKLKLMLNVFKEEAEGII IPGKEDPVKFSH GKLDKKEIESFCLTTKNTEDITKVIYFLCKFLDGKEINELCCAMMNKLDGISDLIETAKQCGEDVEFVDQFKCLSKCATMSNQI RIVKNI SRMKKEMTIDNDTI FLDALELLGRKIEKYQKDKNGDYVKDEKGKKVYTKDYNNFQDMFFEGKNHRVRNFVSNNVIKSK WFSYWRYNKPAECQALMRNSKLVKFALDELPDSQIEKYYISVFGEKSSSSNEEMRRELLKKLCDFSVRGFLDEIVLLSEDEMK QKDKFSEKEKKKSLIRLYLTIVYLITKSMVKINTRFSIACATYERDYILLCQSEKAERAWEKGATAFALTRKFLNHDKPTFEQY YTREREISAMPQEKRKELRKENDQLLKKTHYSKHAYCYIVDNWNLTGAVANDNGRGLPCLSEKNDNANLFLEMRNKIVHLNW HDMVKYINEIKNITSYYAFFCYVLQRMI IGNNSNEQNKFKAKYSKTLQEFGTYSKDLMWVLNLPFAYNLPRYKNLSNEQLFYDE EERMEKIVGRKNDSR (SEQ ID NO: 270)
>OHCW01000317_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPVAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYYMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKWISLLIDMMKGYEPDDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DVAAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMNFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNRIAHLTWRELKEYIGD IRTVDSYFSIYHYVMQRCITKREDDTKQGEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 271)
>OHDP01000241_4
[human gut metagenome]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVPKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVENTVYMTSFGR GNDAVLEQKIVDTSHEQLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEDGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKRLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIKFENFSDAGNYQKANVTSRTSEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNFPGKDDSDEQK (SEQ ID NO: 272)
>OHDT01000502_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEWIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTASYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 273)
>OHFA01000290_5
[human gut metagenorae]
MGKKIHARDLREQRKTDRTVKFADQNKKREAERAVQKKDAAVSVKSVPSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVIGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFDDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDVDNIKNLSGSNIEIRDNELRKCFISYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEDSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPENTALCSANKKREKLADMIAEIEFENFSDAGNYQKANVTSKTHEAEIKRKNQSI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIKEIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDEQK (SEQ ID NO: 274)
>OHGX01000264_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRDTLDYLVDERFDSINKGF VQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDIAAGEALVRKLRFSMTDDEKEGLYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDNRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 275)
>OHIB01002708_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEWIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTASYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNI SFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVISIMQTAA (SEQ ID NO: 276)
>OHJK01001285_9
[human gut metagenome]
MGKKIHARDLREQRKNDRTAKFAVQNKKCEAQRAVQKKDAAVSAKSVSSVSSKKDNATKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAVYALNNTIADENDENWDLFANFSTDNTYYELRNAAAYKESADDESTDDEKRREA EKKKREAKKAEKILADYEKFRKNNRLAYFADAFYVDKNKSKSKSKDKAEGIQRGKKEIYSILALIAKLRHWCVHSEDGRAEFWL YKLDELKDDFKNVLDWYNRPVEEINNRFIENNKVNIQILDSVYENTDIAELTRSYYEFLITKKYKNMGFSIKKLRESMLEGKG YADKEYDSVRNKLYQMTDFILYTGYINEDSDRADDLVNTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKKLS KSNIEIQEDKLRKCFI SYADSVSEFTKLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKY LAELVELNSFVKSCSFDMSAKRTMYRDALDILGIESDKTEEDIEKMIDNILQVDANGKKLPNKNHGLRNFIASNVIDSNRFEYL VRYGNPKKIRETAKCEPAVRFVLNEI PDAQIERYYKAYYPDEKSLCLANMQRDKLADMIAEIKFENFSDAGSYQEANATSTRIT SEAEIKRKNQAI IRLYLTVMYIMLKNLVNVNARYVIAFHCLERDAKLYSESVLKVGNTNEESRLQTGNTNEEKNKVKLTNLTNL TMAVMGVKLENGTIKTEFDKSLAENAANRYLRNARWYKLILDNLKKSERAWTEFRNTVCHLNAIRNININIKEVKEVENYFAL YHYLIQKHLEKRFADKKVERDTGDFI SKLEEHKTYCKDFVKAYCTPFGYNLVRYKNLTIDGLFDKNYPGKDDSDEQK (SEQ ID NO: 277) >OHJS01001864_3
[human gut metagenorae]
MAKKNKMKPRERREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVDEWITFSSKHGFESGVKINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGLENESNNDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTKR LGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRETLDYLIDERFDSINKGFIQGN KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDW AGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC VEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG LYKEII PELASKNLKNDYRILSQTLCELCDNRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGD IRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 278)
>OHJT01001977_4
[human gut metagenome]
MGKKIHARDLRERRKTDRTEKFADQNKKREAERAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFDDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIESSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKARNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKRLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIEFENFSDAGNYQKANVTSRTSEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKIENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 279)
>OHMF01000395_24
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAVEINNNAVPEIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKNSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND VIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 280)
>OHUY01000263_2
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNNYNKTQLSSKDNSNIELGDVDEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGFEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSQMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 281)
>OIBN01003740_1
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKDNSNIQLGGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIED NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLRDKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDI AAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISGILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIC TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 282)
>OIEE01000042_11
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDWEWITFSSKHGFGSGMKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 283)
>OIEL01000292_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRDTLDYLVDERFDSINKGF VQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEVLVRKLRFSMTDDEKEWIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 284)
>OIEN01002196_3
[human gut metagenome]
MERQKRKMKSKSKMAGVKSVFVIGDELLMTSFGDGDDAVLEKDIDENGWNDCRNPAAYDAVYGTDSIRVKKTNNNIRAKWNP LAKSNIRSEESALFRTRVNEYKREQKDKYETLFFGKTFDDNIHIQLISKILDIEKTFSWIGNIVYAINNLSLEQSIDRPIDIF GDKNTQGI SLREDNDYLKTMLPRCEYLFHNILNSDSDNNSKMNYNKWKGKEEKDNRNNENIEKLKKALEVIKI IRVDSFHGVD GIKGDQKFPRSKYNLAWYNEEIQKTISEPFNRKVEEVQQDFYRNSCVNIDFLKEIMYGSNYTDRGSDSLECSYFNFAILKQNK NMGFSITSIRECLLDLYELNFESMQNLRPPANSFCDFLIYDYYCKNESERANLVDCLRSAASEEEKKNIYFQTAERVKEKFRNA FNRISRFDASYIKNSREKNLSGGSSLPKYSFIEGFTKRSKKINDNDEKNADLFCNMLYYLAQFLDGKEINIFLTSIHNIFQNID SFLKVMKEKGMECKFQKDFKMFSHAGHVAKKIEIVI SLAKMKKTLDFYNAQALKDAVTILGVSKKHQYLDMNSYLDFYMFDNRS GATGKNAGKDHNLRNFLVSNVIRSRKFNYLSRYSNLAEVKKLAQNPSLVQFVLSRIEPSLICRYYESSQGISSEGITIDEQIKK LTGI IVDMNIDSFENINNGEIGMRYSKATPQSIERRNQMRVCVGLYLNVLYQIEKNLMNVNARYVLAFAFAERDALMLNFTLEE CKKNKKRSSGGFSFIEMTQFFIDKKLFKVATEAIKKNVLKYNGNPESLNHIPGEYICKNMEGYHENTVRNFRNMVAHLTAVARV PLYI SEVTQIDSYYALYHYCMQMNILQGIEQSGKILDNIKLKNALENARVHRTYSKDAVKYLCLPFAYNI SRYKALTIKDLFDW TEYSCKKDE (SEQ ID NO: 285)
>OIXA01002812_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSKDNSNIQLGGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNINKVKCNIKKSFSTF NDLLKTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSI NKGFIQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFC NYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLY FSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKL TMFRDALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQ IERYYKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAI HCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWR ELKEYIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNE YLTEK (SEQ ID NO: 286)
>OIXU01000818_5
[human gut metagenorae]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNTPKPNKEIKKSKAKLAGVKWVIKANNDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRIPYLKYGGGKDSKGNPKNKSKWKKRENFINFYNEAK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNEFVKNKFKNVNKNFISNEKNNL YIILNAYGEDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIENGYCPLPYDKENDVAKLSSVKHKLYKTYDFVITHY LNSNDKPLLEIVEALRLSKNDDEKEIVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFREKVRINKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFIAENDDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP NEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENYKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGFFYKDICEKKDKKQLYKDDDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDEDRKEYNKFFKQYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFDYCKKNNNWLAKMKDLAHIKSDCDEFSSKHTYPYSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQWLNHYENLNDFIRILY (SEQ ID NO: 287)
>OIXU01000818_6
[human gut metagenome]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNTPKPNKEIKKSKAKLAGVKWVIKANNDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRIPYLKYGGGKDSKGNPKNKSKWKKRENFINFYNEAK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNEFVKNKFKNVNKNFISNEKNNL YIILNAYGEDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIENGYCPLPYDKENDVAKLSSVKHKLYKTYDFVITHY LNSNDKPLLEIVEALRLSKNDDEKEIVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFREKVRINKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFIAENDDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP NEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENYKI PNVEKEKKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGFFYKDICEKKDKKQLYKDDDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDEDRKEYNKFFKQYRNNIVHL NIIAKLSELTKNIDKDINSYFDIYHYCTQRVMFDYCKKNNNWLAKMKDLAHIKSDCDEFSSKHTYPYSSAVLRFMNLPFAYNV PRFKNLSYKKFFDKQ (SEQ ID NO: 288)
>OIYU01000175_4
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELCDVGKVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLK TKRLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFI QGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRD ALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGD IRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 289)
>OIZA01000315_9
[human gut metagenome]
MAKKKRMTAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNKPKPNKEIKKSKAKLAGVKWIIKANDDVTYISSF GKGNNSVLEKRI IGDVSGDVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEIDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDLNYRVPYLEYGGGKDSNGKPNKISAWKKRENFINFYNEAK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNEFVKNKFKNVNKNFISNEKNNL YIILKAYGEDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKLLLEIVETLRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFAEKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFIFHNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP KEPLLKDALLALGVSSNDFDEKYEKYFKTDVDADKDHQKVSTFLMNNVINNSRFKYWKYINPADINGLAKNRYLVKFVLSKIP EEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALYFV ERDGFFYKQICEKKLIETLKKKDKKQLYNDDDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREEYNKFFKQYR NNIVHLKI IAKLSELTKNIDKDINSYFDIYHYCTQRVMFDYCKKNNNWLAKMKDLAHIKSDCDEFSSKHTYPYSSAVLRFMNL PFAYNVPRFKNLSYKKFFDKQ (SEQ ID NO: 290)
>OIZI01000180_12
[human gut metagenorae]
MAKKKRMTAKERKQNHRDSLMKKADSNAEKEKAKKPWENKPDTAISKDNKPKPNKEIKKSKAKLAGVKWIIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSLDRSYNQSGNDTIGFDLNYCIPYSEYGGGKDSNGKPNKISAWKKRENFIKFYNEAK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNEFVKNKFKNVNKNFISNEKNNL YIILNAYGEDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIENGYCPLPYDKENDVAKLSSVKHKLYKTYDFVITHY LNSNDKPLLEIVEALRLSKNDDEKEIVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFREKVRINKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFIAENDDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP NEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKADKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEHSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLWINGLYTLALY FVERDGFFYKQICEKKLIETLKKKDKKQLYKDDDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREDYNDFFTA YRNNIVHLNI IAKLSKLTKNIDKDINSYFDIYHYCTQRVMFDYCKKNNNWLAKMKDLAHIKSDCDEFSSKHTYPYSSAVLRFM NLPFAYNVPRFKNLSYKKFFDKQWLNHYENLNDFIRILY (SEQ ID NO: 291)
>OIZI01000180_12
[human gut metagenome]
MAKKKRMTAKERKQNHRDSLMKKADSNAEKEKAKKPWENKPDTAISKDNKPKPNKEIKKSKAKLAGVKWIIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSLDRSYNQSGNDTIGFDLNYCIPYSEYGGGKDSNGKPNKISAWKKRENFIKFYNEAK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDNDIELYSENYSEEYVFINCLNEFVKNKFKNVNKNFISNEKNNL YIILNAYGEDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIENGYCPLPYDKENDVAKLSSVKHKLYKTYDFVITHY LNSNDKPLLEIVEALRLSKNDDEKEIVYKKYAEKLFKADDVINPIKAI SKLFVEKGNKLFREKVRINKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQI IEDHNNVIKFIAENDDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP NEPLLKDALLSLGVSDDTKVLENTYKKYFDSKEKADKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSK IPEEQIDSYYKLFSNEEEHSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALY FVERDGFFYKQICEKKLIETLKKKDKKQLYKDDDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREDYNDFFTA YRNNIVHLNI IAKLSKLTKNIDKDINSYFDIYHYCTQRVMFDYCKKNNNWLAKMKDLAHIKSDCDEFSSKHTYPYSSAVLRFM NLPFAYNVPRFKNLSYKKFFDKQ (SEQ ID NO: 292)
>OIZU01000200_48
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLNYLVDERFDSIN KGFIQGNKVNISLLIDMMKDDYEADDIIHLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFC NYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLY FSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKL TMFRDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQ IERYYKSCVEFPDMNSSLKVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAI HCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWR ELKEYIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNE YLTEK (SEQ ID NO: 293)
>OIZW01000344_20
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIED NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VADEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEVSDMNSSLEAKRSELARMIKNIRFDDFKNVKQQANGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSSMIRKYRNCIAHLTWRELNKYIN DIYVWSYFSICHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIKQLFDRNEYLTEK (SEQ ID NO: 294)
>OIZX01000427_25
[human gut metagenorae]
MAKKKKTARQLREEMQQQRKQAIQKQQEQRQEKAAAARETAAPEQPAAAPVPKRQRKSLAKAAGLKSNFILDPQRRTTVMTAFG QGSTAILEKQIVDRAI SDLQPVQQFQVEPASAAKYRLKNSRVRFPNVTADDPLYRRKDGGFVPGMDALRRKNVLEQRFFGKSFA DNIHIQMIYSILDIHKILAAASGHIVHLLNIVNGSKDRDFIGMLAAHVLYNELNEEAKRSIADFCKSPRLIYYSAAFYETLDNG KSERRSNEDI FNILALMTCLRNFSSHHSIAIKVKDYSAAGLYNLRRLGPDMKKMLDTFYTEAFIQLNQSFQDHNTTNLTCLFDI LNISDSARQKQLAEEFYRYWFKEQKNLGFSVRKLREEMLLLPDAAVIADKRYDTCRSKLYNLMDFLILRVYRTGRADRCDKLP EALPAALTDEEKAWYHKEALSLWNEMRTLILDGLLPQMTPENLSRLSGQKRKGELSLDDAMLKECLYEPGPVPEDAAPEEANA EYFCRMIYLATLFMDGKEINTLLTTLISKFENIAAFLQTMEQLNIEAELGPEYAMFTRSRAVAEQLRVINSFALMKKPQVNAKQ QLYPAAVTLLGTEDPDGVTDEMLCIDPVTGKMLPPNQRHHGDTGLRNFIANNWESRRFQYLIRYSDPAQLHQLASNKKLVRFV LSSIPDTQINRYYETCGQTRLAGRAAKVEFLTDMIAAIRFDQFRDVNQKERGANTQKERYKAMLGLYQTVLYLAVKNLWINAR YVMAFHCVERDMFLYDGELTDPKGESVSAFLAWGKKGVQPQYLLLTQLFIRRDYLKRSACEQIQHNMENISDRLLREYRNAVA HLNVIAHLADYSADMREITSYYGLYHYLMQRHLFKRHAWQIRQPERPTEEEQKLIEQEQKQLAWEKALFDKTLQYHSYNKDLVK ALNAPFGYNLARYKNLSIEPLFSKEAAPAAEIKATHA (SEQ ID NO: 295)
>OIZX01000427_26
[human gut metagenome]
MLLSEELYKWGKAGSTMAKKKKTARQLREEMQQQRKQAIQKQQEQRQEKAAAARETAAPEQPAAAPVPKRQRKSLAKAAGLKSN FILDPQRRTTVMTAFGQGSTAILEKQIVDRAI SDLQPVQQFQVEPASAAKYRLKNSRVRFPNVTADDPLYRRKDGGFVPGMDAL RRKNVLEQRFFGKSFADNIHIQMIYSILDIHKILAAASGHIVHLLNIVNGSKDRDFIGMLAAHVLYNELNEEAKRSIADFCKSP RLIYYSAAFYETLDNGKSERRSNEDI FNILALMTCLRNFSSHHSIAIKVKDYSAAGLYNLRRLGPDMKKMLDTFYTEAFIQLNQ SFQDHNTTNLTCLFDILNISDSARQKQLAEEFYRYWFKEQKNLGFSVRKLREEMLLLPDAAVIADKRYDTCRSKLYNLMDFLI LRVYRTGRADRCDKLPEALPAALTDEEKAWYHKEALSLWNEMRTLILDGLLPQMTPENLSRLSGQKRKGELSLDDAMLKECLY EPGPVPEDAAPEEANAEYFCRMIYLATLFMDGKEINTLLTTLISKFENIAAFLQTMEQLNIEAELGPEYAMFTRSRAVAEQLRV INSFALMKKPQVNAKQQLYPAAVTLLGTEDPDGVTDEMLCIDPVTGKMLPPNQRHHGDTGLRNFIANNWESRRFQYLIRYSDP AQLHQLASNKKLVRFVLSSI PDTQINRYYETCGQTRIA.GRAAKVEFLTDMIAAIRFDQFRDVNQKERGANTQKERYKAMLGLYQ TVLYLAVKNLWINARYVMAFHCVERDMFLYDGELTDPKGESVSAFLAWGKKGVQPQYLLLTQLFIRRDYLKRSACEQIQHNM ENISDRLLREYRNAVAHLNVIAHLADYSADMREITSYYGLYHYLMQRHLFKRHAWQIRQPERPTEEEQKLIEQEQKQLAWEKAL FDKTLQYHSYNKDLVKALNAPFGYNLARYKNLSIEPLFSKEAAPAAEIKATHA (SEQ ID NO: 296)
>OJMJ01002228_5
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELRGVTKWITFSSKHGLESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKDFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDILYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDTYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 297)
>OJMM01002900_7
[human gut metagenome]
MMGKHLNAKQRKLEKKLKNQQKDMMYTKSTDAVSVPTLKAAPTKAEMSQDTAEASTLITPGTLKTKAKAMGLKSTLVFDDKIW TSFLNSKTEENEKCAHIEKIADCNGQTIVERPRMFNTSINAKKVDLSKDNDETNYPNPAFEDCGRDYINVKSALEKRVFGKTYN KDNLHVQIAYNI FDIKKI IGAYINNI IYIFYNLGREEYDAKKDI IGTQDSAYISKILNNTSAYFTYFDGVFKQITDRDSNKDRE IKNSYNALVLKVLYYLRQFCMHGNTYTKRNEESFLSDTALYNAKEFFAKADPQINELIDAVYADGIKTINSDFMAHAKNNMYII CEVYKNEAEDSLMKEYYDFWRKEGNNLGFNTRQLREILIDKYVGNLRGKKYNTFRNKLYTVLGFILVKEIKRNPKIQDSFIAK LRANQNGDEGKLNIYNEFAPKIWSWSSKLNSAITCFDEESLSKFKGYKDIDESLI SRYGITVANTDTLVKILYFLCKFLDGKE INELCCAMINKFDNINDLIKTAAQCGEDIEFVKEYKLFINSNDLSDQIRIVKSI SKMKPELSKIGEALILDAIDILGYKINKYK YDAAGNRLVDSNNKPVYSEEYCAFKKDFFETCELDEFGRVKYNKKGKPVINHRRRNFI INNVLSSKWFFYVAKYNRPSECQKFM KSKKLIALVLKDVPETQIARYYQSVTGGRTQANSEAMRMTLIKLLHEFSIKNVLSDVGTMTASENKRQIENSRKERMKAIVKLY LTWYLIAKSLVKVNTRFSIAFSAYERDVSLLADENELIALANNEDDKWKKGNYVFALTKHFWDNDEPYFDKYNNALQQIRSIV DPNERRIA.YRANDKVVKHTHFNLHSYKYVKHNYEEI SKASKI ITAYRNNVQHLNVMNSITKYLGDI SEVTSYYSLYCYTLQRLL LDDNNNDKFASIKGNLRKFGIYNKDFMWLLNI PFAYNLPRYKNLSNEEIFYDELQK (SEQ ID NO: 298) >OJMM01002900_7
[human gut metagenorae]
MGKHLNAKQRKLEKKLKNQQKDMMYTKSTDAVSVPTLKAAPTKAEMSQDTAEASTLITPGTLKTKAKAMGLKSTLVFDDKIWT SFLNSKTEENEKCAHIEKIADCNGQTIVERPRMFNTSINAKKVDLSKDNDETNYPNPAFEDCGRDYINVKSALEKRVFGKTYNK DNLHVQIAYNIFDIKKIIGAYINNIIYIFYNLGREEYDAKKDIIGTQDSAYISKILNNTSAYFTYFDGVFKQITDRDSNKDREI KNSYNALVLKVLYYLRQFCMHGNTYTKRNEESFLSDTALYNAKEFFAKADPQINELIDAVYADGIKTINSDFMAHAKNNMYIIC EVYKNEAEDSLMKEYYDFWRKEGNNLGFNTRQLREILIDKYVGNLRGKKYNTFRNKLYTVLGFILVKEIKRNPKIQDSFIAKL RANQNGDEGKLNIYNEFAPKIWSWSSKLNSAITCFDEESLSKFKGYKDIDESLISRYGITVANTDTLVKILYFLCKFLDGKEI NELCCAMINKFDNINDLIKTAAQCGEDIEFVKEYKLFINSNDLSDQIRIVKSISKMKPELSKIGEALILDAIDILGYKINKYKY DAAGNRLVDSNNKPVYSEEYCAFKKDFFETCELDEFGRVKYNKKGKPVINHRRRNFIINNVLSSKWFFYVAKYNRPSECQKFMK SKKLIALVLKDVPETQIARYYQSVTGGRTQANSEAMRMTLIKLLHEFSIKNVLSDVGTMTASENKRQIENSRKERMKAIVKLYL TWYLIAKSLVKWTRFSIAFSAYERDVSLLADENELIALANNEDDKWKKGNYVFALTKHF DNDEPYFDKYNNALQQIRSIVD PNERRLAYRANDKWKHTHFNLHSYKYVKHNYEEISKASKIITAYRNNVQHLNVMNSITKYLGDISEVTSYYSLYCYTLQRLLL DDNNNDKFASIKGNLRKFGIYNKDFMWLLNIPFAYNLPRYKNLSNEEI FYDELQK (SEQ ID NO: 299)
>OJMN01000417_22
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKVAEINNNAAPAIAAMPAVEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTNPNGSTLSDDKKENIRKSLSKFNALLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND WAGEALVRKLRFSMTDDEKEGTYADEAEKLWGKFRNDFENIADHMNGDAIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDI RTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 300)
>OJNR01001167_9
[human gut metagenome]
MGKKIHARDLREQRKTDRTVKFADQNKKREAQRAVQKKDAAVSVKSVSSVSSKKDNATKSMAKAAGVKSVFAVENTVYMTSFGR GNDAVLEQKIVDTSHEQLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKARNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDVDNIKNLSGSNIEIRDNELRKCFISYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIESDKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCEPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIEFENFSDAGNYQKANVTSRTSEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNSPGKDDSDEQK (SEQ ID NO: 301)
>OJPG01000139_73
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKRGNESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIA
AGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEIIPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIRT
VDSYFSIYHYVMQRCITKREDDIKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 302)
>OJPX01000614_4
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKVAEINNNAAPAIAAMPAVEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTNPNGSTLSDDKKENIRKSLSKFNALLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFVQG NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND WAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEVKRSELARMIKNICFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEIVSELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDI RAVDSYFSIYHYVMQRCITKRGNDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 303)
>OKRZ01002949_5
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSIFVSENKMYITSFGKGNSAVLE YEVDKVDNNVYNQTQLSSEDSSNIELCGVTKVNITFSSKHGLESGVEI STSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQFIYNILDIEKILAVYVTNSVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDKSSKLDEDLYSFIDI IDPEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 304)
>OKSB01002689_10
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDIYSFINNIDPEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGETLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTEGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDVLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 305)
>OKSC01004083_2
[human gut metagenome]
MEREVKKPPKKSLAKAAGLKSTFVISPQEKELAMTAFGRGNDALLQKRIVDGWRDVAGEKQQFQVQRQDESRFRLQNSRLADR TVTADDPLHRAETPRRQPLGAGMDQLRRKAILEQKYFGRTFDDNIHIQLIYNILDIHKMLAVPANHIVHTLNLLGGYGETDFVG MLPAGLPYDKLRWKKKNGDTVDIKADIAAYAKRPQLAYLGAAFYDVTPGKSKRDAARGRVKREQDVYTILSLMSLLRQFCAHD SVRIWGQNTPAALYHLQALPQDMKDLLDDGWRRALGGVNDHFLDTNKVNLLTLFEYYGAETKQARVALTQDFYRFWLKEQKNM GFSLRRLREELLKLPDAAYLTGQEYDSVRQKLYMLLDFLLCRLYAQERTGRCEELVSALRCALSDEEKDAVYQAEAAALWQALG DTLRRELLPLLKGKKLQDKDKKKLDELGLSRDVLDGVLFRPAQQGNPANADYFCRLMHLSTWFMDGKEINTLLTTLISKLENID SLRNVLESMGIA.CSFVPAYAMFDHSRYIAGQLRVWNIARMRKPAITAKREMYRAAVVLLGVDSPEAAAAITDDLLQIDPETGK VRPRGDSARDTGLRNFIANNWESRRFTYLLRYMTPEQARVLAQNEKLIAFVLSTVPDTQLERYCRTCGREDITGRPAQIRYLT AQIMGVRYESFTDVEQRGRGDNPKKERYKALIDLYLTVLYLAVKNMVNCNARYVIAFYCRDRDTALYQKEVCWYDLEEDKKSGK QRQVEDYTALTRYFVSQGYLNRHACGYLRSNMNGISNSLLTAYRNAVDHLNVIPPLGSLCRDIGRVDSYFALYHYAVQRYLNGR YYRKTPREQELFAAMAQHRTWCSDLVKALNTPFGYNLARYKNLSIDGLFDREGDHWREDGEKPAE (SEQ ID NO: 306)
>OKSD01002505_11
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAEEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDKVDNDNYNKTQLSSEDSSNIELCGVTKVNITFSSKHGLESGVEI STSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQFIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDI IDPEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 307)
>OLGN01000304_32
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDSSNIELRGVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESHDDFMGYLSAKNTYDVFTNPNGSTLSDDKKKNIRKSLRKFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKDDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND VIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDI RTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 308)
>OLHE01000257_41
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKVAEINNNAAPAIAAMPAAEAAAPAVEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVDEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVNERFDSINKGFIQG NKWISLLIDMMKGDYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRND WAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNSDVIKQLGKADMDFDEKILDSEKKNASDLLYFSKMIY MLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDA LTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYK SCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERD FGLYKEII PELASKNLKNDYRILSQTLCELCDNCDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 309)
>PPYE01106492_34
[human gut metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVDEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGESLVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFWKNIASMRKPAVSAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 310)
>PPYE01385196_3
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKVAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKNSSNIELCGVTKVNITFSSKHGFGSGVKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFN DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSARNTYKVFTRPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDPEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDVAAGEALVRKLRFSMTDDEKEGIYADEAEKLWVKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYLSKM IYMLTYFLDGKEINDILTTLISKFDNIKEFLKIMKSSAWVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNFPRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 311)
>PPYE01512733_3
[human gut metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMSAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAKNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGETLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 312)
>PPYF01670242_39
[human gut metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYITNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNALLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVIKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 313)
>ODFW01000112_43
[human metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDNGDESPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 314)
>ODGN01000188_50
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSDGSSNIELRGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFVQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 315)
>ODHH01000275_14
[human metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMSAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEWITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAKNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVEERLKSINKDFIEG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDPVRSKMYKLMDFLLFCNYYRNDV VTGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYMILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSSFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 316)
>ODHP01001712_3
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDW
AGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDALT
ILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEIIPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIRT
VDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 317)
>ODHV01000466_16
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEASKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKCSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 318)
>ODJZ01000182_13
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE
YEVDNNDYNKTQLSSKDNSNIELCDVGKWITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKNFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTPASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDW
AGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGD
IRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 319) >ODLN01002572_7
[human metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 320)
>ODQJ01000729_25
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKGSSNIELHGVNEINITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSAKNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLGVKRSELARMIKNISFDDFKNVKQQSKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIC TVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 321)
>ODUN01000242_23
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVDEVNITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQFIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFIGYLSARNTYKVFTHPDKSNLSDKAKGNIKKSFSTFNDLLKTK RLGYFGLEEPKTKDTRVLEAYKKRVYYMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWVKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 322)
>ODVQ01003982_3
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDSSNIELRGVNEVNITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESHDDFMGYLSAKNTYDVFTNPNGSTLSDDKKKNIRKSLRKFNDLLKTK RLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLHEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV AAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKI IDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPASSAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIRKVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 323)
>ODVR01002077_3
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDSSNIELHGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDIIDSEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEVPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDKSSNLFLKKNKRLRKCVEVDINNADSRMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKREEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 324)
>ODXC01000747_3
[human metagenorae]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKGSSNIELHGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDDESHDDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRETLDYLIDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGESLVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFWKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 325)
>ODXO01005124_2
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAVEINNNAVPEIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCDVDEWITFSSKHGFESGVKINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKDSESYDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSLSKFNALLKTK RLGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDI AAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKVWiFVLGGIPDTQIERYYKS CVEFPDMNSSLKVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTWiYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNRRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 326)
>ODYC01000377_16
[human metagenome]
MAKKNKMKPRELREAQKKARQFKAAEINNNAAPAIAAMPAAEVIAPVAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELCGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR LGYFGLEEPKTKDTRVLEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLDYLVDERFDSINKGFI QGNKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKILDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRN DIAAGESLVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMI YMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRD ALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYY KSCVEFPDMNSSLEVKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLER DFGLYKEI IPELASKNLKNDYRILSQTLCELCDNRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEY IGDIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 327)
>OEJW01000623_11
[human metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNQTQLSSDGSSNIELRGVNEWITFSSKHGFESGVEINTSNPTHRSGESSPVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFVQG NKWISLLIDMMKGYEVDDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGESLVRKLRFSMTDDEKEGIYADEAA.KLWGKFRNDFENIADHMNGDVIKELGKADMGFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVNVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLKAKRSELARMIKNISFEDFKDVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQMLCELCDDRDKSPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIYAVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDNLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 328)
>3300019376 I GaOl 87899_10021543_4
[mammals-digestive system- feces]
MNKIHKKQGKTTAKSLGLKSVLKIENDLWTTFGKKDNPMWEQSINKASGEKELYVDEDQVKFDSSLIKEKNILSLDSIQHSN HQIIWIDQKDASEIGMDYLRLKPELEKEFFGKTFYDNVHIQIAYNLLDLKKIIGLHIGNAIQALENLGRDGSDLVGICDATKP LNYLDDVKQKADIGFMNRLKPYFMYFDGVLKLDNSKNKNGELNQLDIENWDVIRILSLIRQGCAHAGAYSSLLYTAQNNKVYAD LINKALSI FSDDLDKFNKSFLKQSKMNLFILFDLYNCRFDRSLQEKIIKEYYRYVLYKDNKNLGFSLKNVRNLI IEGKYDEQER SGKLQTIRSKLNTLLDFYLYGYYQKNPTFVENIVAKLRESKNDEDKEKVYEEEYHRLLSENNYLVDKKCSDIVYRINEAVKNRK IFWANINAVVEKVSCSCFPSLIYVLCKFLDGKEVNELTTAI INKLENIASLINALVTLKSYGGFSEQYKIFDYPNINGLIDDF RMVKNLTSTKRKLKKASGGEDRIGRQLYADAINI FKEDSFVSANDEKGTGLDQYWKFFSKDDLGARKVRNLLLNNIIKNRRFV YLIKYIDPKDCYKLVHNEKIVRFALGQYDESQMPLNQLQKYYDAVIENREGFRKCNDRKKIIDTLVSEINRVSIDGILDIGNRL WRGNNDYINHQKQII SLYLTIAYLIVKGVVHTNSLYFIAWHAYERDNNFKFGNDGKDYLALTKEYLTNKKKRVKQLLDHNIEE ANNSLDSKYFSAYRNKWHLNFCNIFWYLDGIGDIHSYYDIYQYVIQKWSIAERSKDFIDPQYLTKLSNDLKQYRTYQRNFLK IINLPFAYNLARYKNLTIGDLFNDKYPLPKETVKEFYNEE (SEQ ID NO: 329)
>OGCZ01001955_1
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAQVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKGSESYDDFMGYLSARNTYEVFTHPDKSNLSDKVKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLNYLVDERFDSIN KGFIQGNKWISLLIDMMKDDYEADDIIHLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFC NYYRNDWAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLY FSKMIYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYRLFNDSQRITNELFIVKNIASMRKPAASAKL TMFRDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQ IERYYKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAI HCLERDFGLYKEII PELASKNLKNDYRILSQTLCELCDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELK EYIGDIRTVDSYFSIYHYVMQRCITKREDDKKQEEKIKFEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLT EK (SEQ ID NO: 330)
>OGDY01002059_17
[metagenome]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEQLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLNELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLWTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKRLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPENTALCSANKKREKLADMIAEIEFENFSDAGNYQKANVTSKTHEAEIKRKNQSI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNINIKEIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPFGY NLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 331)
>OGEU01000713_24
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDDNDYNKTQLSSKDNSNIELGNWEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDI IDSEYRDTLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKIMDFLLFCNYY RNDVVAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMF RDALAILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEVPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCL ERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYI GDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 332)
>OGFM01002125_3
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEAAAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNDYNQTQLSSKGSSNIELHGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKDSESYDDFMGYLSAKNTYDVFTDPDESDLSKNIKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSEAYKKRVYHMLAIVGQIRQCVFHDLSEHSEYDLYSFIDNSKKVYRECRETLNYLVDERFDSIN KGFIQGNKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCN YYRNDVVAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYF SKMIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFWKNIASMRKPAASAKLT MFRDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQI ERYYKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIH CLERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRE LKEYIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEY LTEK (SEQ ID NO: 333)
>OGHW01002048_1
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSESSSNIELCGVTKVNITFSSKHGFGSGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEVDDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDVAAGEALVRKLRFSMTDDEKEGIYADEAEKLWGKFRNDFENIADHMNGDAIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSYAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQKLCELCDKSPNLFLKKNERLRKCVEVDINNADSIMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 334)
>OGIE01002059_21
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKRGNESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYETDDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGTYADEAEKLWGKFRNDFDNIAGHMNGDAIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRTLSQTLCGLCDKSPNLFLKKNKRLRKCVEVDINNADSIMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 335)
>OGJI01000038_151
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNDDYNKTQLSSKGSSNIELHGVNEVNITFSSKHGFESGVEI STSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQFIYNILDIEKILAVYVTNIVYALNNMLGVKDSESYDDFMGYLSARNTYKVFTHPDKSNLSDKVKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQCVFHDKSSKLHEDLYSFINNIDPEYRDTLDYLVEERLKSINKDF IEGNKVNI SLLIDMMKDDYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYY RNDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSK MIYMLTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECKLTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMF RDALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIER YYKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCL ERDFGLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELK EYIGDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLT EK (SEQ ID NO: 336)
>OGJK01007642_2
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNNYNKTQLSSKDNSNIELGDWEVNITFSSKRGNESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDVIAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDAIKELGKADMDFDEKILDSEKKYASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDERDKSPNLFLKKNERLRKCVEVDINNADSIMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 337)
>OGJY01000516_18
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPVAGKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKVDNNDYNQTQLSSKGSSNIELCGWEVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGIKKSESYDDFMGYLSARNTYEVFTHPDKSNLSDKAKGNIKKSFSTFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFIDI IDSEYRETLDYLVEERLKSINKDF IEGNKVNI SLLIDMMKGFEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGYRFKDKQYDSVCSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEVKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDKSSNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEDKIKYEDNLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 338)
>OGKA01000617_2
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAVPAIAAMPAVEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYIFTHPDKSNLSDKVKGNIKKSLSKFNDLLKTK RLGYFGLEEPKTKDKRVSEAYKKRVYHMLAIVGQIRQSVFHDKSNELDEYLYSFIDIIDSEYRDTLDYLVDERFDSINKGFIQG NKVNISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFKNDFENIADHMNGDVIKEFGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIGDIR TVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK (SEQ ID NO: 339)
>OGKG01002483_14
[metagenome]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYVNKKNPKGKARNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLDELKDDFKNVLDWYNRPVEEIN NRFIENNKVNIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLVNTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDVDNIKNLSGSNIEIRDNELRKCFISYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEILDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIKFENFSDAGNYQKANVTSRTSEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVIGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAWNEFRNTVCHLNAIRNININIKEIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 340) >OGKW01000585_4
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNATPTIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE
YEVDNNDYNQTQLSSKNSSNIELRGVNEVNITFSSKHGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNALLKTKR
LGYFGLEEPKTKDTRASEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRETLDYLVDERFDSINKGFIQGN
KVNISLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKIMDFLLFCNYYRNDW
AGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAWVECELTVGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEII PELVSKNLKNDYRILSQTLCELCDKSPNLFLKKNERLRKCVEVDINNADSVMTRKYRNCIAHLTVVRELKEYIGDIRT
VDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK (SEQ
ID NO: 341)
>OGLJ01000192_54
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAVEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDKMDNNNYNKTQLSSESSSNIKLCGVTKVNITFSSKHGFESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNTYDVFIDPDNSSLSDDKKANVRKSLSKFNALL KTKRLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIYPEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDVAAGEALVRKLRFSMTDDEKEGIYAGEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFR DALTILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERY YKSCVEVPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDKSPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKRENDTKQEDKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 342)
>OGLM01001314_21
[metagenome]
MGKKIHARDLREQRKTDRTEKFADQNKKREAERAVQKKDAAVSVKSVSSVSSKKDNVTKSMAKAAGVKSVFAVGNTVYMTSFGR GNDAVLEQKIVDTSHEPLNIDDPAYQLNWTMNGYSVTGHRGETVSAVTDNPLRRFNGGKKDEPEQSVPTDMLCLKPTLEKKFF GKEFNDNIHIQLIYNILDIEKILAVYSTNAIYALNNMSADENIENSDFFMKRTTDETFDDFEKKKESTNSREKADFDAFEKFIG NYRLAYFADAFYWKKNPKGKAKNVLREDKELYSVLTLIGKLRHWCVHSEEGRAEFWLYKLNELKDDFKNVLDWYNRPVEEIN NRFIENNKWIQILGSVYKNTDIAELVRSYYEFLITKKYKNMGFSIKKLRESMLEGKGYADKEYDSVRNKLYQMTDFILYTGYI NEDSDRADDLVNTLRSSLKEDDKTTVYCKEADYLWKKYRESIREVADALDGDNIKRLSKSNIEIQEDKLRKCFI SYADSVSEFT KLIYLLTRFLSGKEINDLVTTLINKFDNIRSFLEIMDELGLDRTFTAEYSFFEGSTKYLAELVELNSFVKSCSFDINAKRTMYR DALDILGIKSGKTEEDIEKMIDNILQIDANGDKKLKKNNGLRNFIASNVIDSNRFKYLVRYGNPKKIRETAKCKPAVRFVLNEI PDAQIERYYEACCPKNTALCSANKRREKLADMIAEIKFENFSDAGNYQKANVTSKTHEAEIKRKNQAI IRLYLTVMYIMLKNLV NVNARYVIAFHCVERDTKLYAESGLEVGNIEKNKTNLTMAVMGVKLENGI IKTEFDKSLAENAANRYLRNARWYKLILDNLKKS ERAVWEFRNTVCHLNAIRNININIDGIKEVENYFALYHYLIQKHLENRFADKKVERDTGDFISKLEEHKTYCKDFVKAYCTPF GYNLVRYKNLTIDGLFDKNYPGKDDSDKQK (SEQ ID NO: 343)
>OGMO01000062_69
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSKNKMYITSFGKGNSAVLE YEVDKVDNDNYNKTQLSSEDSSNIELCGVTKVNITFSSKHGLESGVEINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFD DNIHIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESYDDFMGYLSAQNTYYI FTHPDKSNLSDKVKGNIKKSLSKFNDLL KTKRLGYFGLEEPKTKDTRVSQAYKKRVYHMLAIVGQIRQSVFHDKSSKLDEDLYSFINNIDPEYRETLDYLVDERFDSINKGF IQGNKVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLDEYGYRFKDKQYDSVRSKMYKLMDFLLFCNYYR NDWAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKM IYMLTYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTVGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMLR DALTILGIDDKITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAENEKWMFVLGGIPDTQIERY YKSCVEFPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQANGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLE RDFGLYKEII PELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKE YIGDIRTVDSYFSIYHYVMQRCITKREDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTE
K (SEQ ID NO: 344)
>OGMP01001167_15
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGNVNEVNITFSSRRGFESGVEINTSNPTHRSGESSSVRGDMLGLKSELEKRFFGKTFDDNI
HIQLIYNILDIEKILAVYVTNIVYALNNMLGEGDESNYDFMGYLSTFNTYKVFTNPNGSTLSDDKKENIRKSLSKFNVLLKTKR
LGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIQGN
KVNI SLLIDMMKGYEADDIIRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDIA
AGEALVRKLRFSMTDDEKEGIYAGEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYML
TYFLDGKEINDLLTTLISKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDALT
ILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKSC
VEVPDMNSSLEAKRSELARMIKNIRFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLVNVNARYVIAIHCLERDFG
LYKEII PELASKNLKNDYRILSQTLCELCDDRDKSPNLFLKKNKRLRKCVEVDINNADSNMTRKYRNCIAHLTWRELKEYIGD
IRTVDSYFSIYHYVMQRRITKRKDDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNI PRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 345)
>OGUJ01000114_43
[metagenome]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRVPYSEYGGGKDSNGEPKNQSKWKKRKNFIKFYNKSK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKILLEIVEVLRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFAEKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISHNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP QEPLLNDALLALGVSKTDLENTYNKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSKIP EEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALYFV ERDRYFYKKICGKALRRKVGDKYDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREGYNDFFTAYRNNIVHLNI IAKLSELTKNIDKDINSYFDIYHYCTQRVMFDYCKMNNNWLAKMKDLAHIKSDCDEFSSKHTYPFSSAVLRFMNLPFAYNVPR FKNLSYKKFFDKQWLNH (SEQ ID NO: 346)
>OGUJ01000114_45
[metagenome]
MAKKKRITAKERKQNHRESLMKKADSNAEKEKAKKPWENKPDTAISKDNTPKPNKEIKKSKAKLAGVKWVIKANDDVAYISSF GKGNNSVLEKRIMGDVSSNVNKDSHMYVNPKYTKKNYEIKNGFSSGSSLVTYPNKPDKNSGMDALCLKPYFEKDFFGHIFTDNM HIQAIYNI FDIEKILAKHITNI IYTVNSFDRNYNQSGNDTIGFDINYRVPYSEYGGGKDSNGEPKNQSKWKKRKNFIKFYNKSK PHLGYYENIFYDHGEPISEEKFYNYLNILNFIRNNTFHYKDDDIELYSENYSEEFVFINCLNKFVKNKFKNVNKNFISNEKNNL YIILNAYGKDTENVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSVKHKLYKTYDFVITHY LNSNDKILLEIVEVLRLSKNDDEKENVYKKYAEKLFKADDVINPIKAI SKLFAEKGNKLFKEKI IIKKEYIEDVSIDKNIYDFT KVIFFMTCFLDGKEINDLLTNI ISKLQVIEDHNNVIKFISHNKDAVYKDYSDKYAI FRNAGKIATELEAIKSIARMENKIENAP QEPLLNDALLALGVSKTDLENTYNKYFDSKEKTDKQSQKVSTFLMNNVINNNRFKYVIKYINPADINGLAKNRYLVKFVLSKIP EEQIDSYYKLFSNEEEPSCEEKIKLLTKKI SKLNFQTLFENNKI PNVEKERKKAIITLYFTIVYILVKNLVNINGLYTLALYFV ERDRYFYKKICGKALRRKVGDKYDYLLLPEIFSGSKYREETKNLKLPKEKDRDIMKKYLPNDKDREGYNDFFTAYRNNIVHLNI IAKLSELTKNIDKDINSYFDIYHYCTQRVMFDYCKMNNNWLAKMKDLAHIKSDCDEFSSKHTYPFSSAVLRFMNLPFAYNVPR FKNLSYKKFFDKQ (SEQ ID NO: 347)
>OJKY01000879_3
[metagenome]
MAKKITAKQKREEKERLNKQKWAKNDSVIIVPETKEEIKTGEIQDNNRKRSRQKSQAKAMGLKAVLSFDNKIAIASFVSSKNAK SSHIERITDKEGTTISVNSKMFESSVNKRDINIEKRITIEEPQQDGTIKKEEKGVKSTTCNPYFKVGGKDYIGIKEIAEEHFFG RAFPNENLRVQIAYNI FDVQKILGTFVNNI IYSFYNLSRDEVQSDNDVIGMLYSISDYDRQKETETFLQAKSLLKQTEAYYAYF DDVFKKNKKPDKNKEGDNSKQYQENLRHNFNILRVLSFLRQICMHAEVHVSDDEGCARTQNYTDSLEALFNI SKAFGKKMPELK TLIDNIYSKGINAINDEFVKNGKNNLYILSKVYPNEKREVLLREYYNFWCKEGSNIGISTRKLKETMIAQNMPSLKEENTYRN KLYTVMNFILVRELKNCATIREQMIKELRANMDEEEGRDRIYSKYAKEIYLYVKDKLKLMLNVFKEEAEGII IPGKEDPVKFSH GKLDKKEIESFCLTTKNTEDITKVIYFLCKFLDGKEINELCCAMMNKLDGISDLIETAKQCGEDVEFVDQFKCLSKCATMSNQI RIVKNI SRMKKEMTIDNDTI FLDALELLGRKIEKYQKDKNGDYVKDEKGKKVYTKDYNNFQDMFFEGKNHRVRNFVSNNVIKSK WFSYWRYNKPAECQALMRNSKLVKFALDELPDSQIEKYYISVFGEKSSSSNEEMRRELLKKLCDFSVRGFLDEIVLLSEDEMK QKDKFSEKEKKKSLIRLYLTIVYLITKSMVKINTRFSIACATYERDYILLCQSEKAERAWEKGATAFALTRKFLNHDKPTFEQY YTREREISAMPQEKRKELRKENDQLLKKTHYSKHAYCYIVDNVNNLTGAVANDNGRGLPCLSEKNDNANLFVEMRNKIVHLNW HDMVKYINEIKNITSYYAFFCYVLQRMI IGNNSNEQNKFKAKYSKTLQEFGTYSKDLMWVLNLPFAYNLPRYKNLSNEQLFYDE EERMEKIVGRKNDSR (SEQ ID NO: 348)
>OLJF01000187_58
[metagenome]
MAKKNKMKPRELREAQKKARQLKAAEINNNAAPAIAAMPAAEVIAPAAEKKKSSVKAAGMKSILVSENKMYITSFGKGNSAVLE YEVDNNDYNKTQLSSKDNSNIELGDVNEVNITFSSKHGFGSGMKINTSNPTHRSGESSPVRWDMLGLKSELEKRFFGKTFDDNI HIQLIYNILDIEKILAVYVTNIVYALNNMLGVKGSESHDDFIGYLSTNNIYDVFIDPDNSSLSDDKKANVRKSLSKFNVLLKTK RLGYFGLEEPKTKDNRVSEAYKKRVYHMLAIVGQIRQCVFHDKSGAKRFDLYSFINNIDPEYRDTLDYLVEERLKSINKDFIQG NKWISLLIDMMKGYEADDI IRLYYDFIVLKSQKNLGFSIKKLREKMLEEYGFRFKDKQYDSVRSKMYKLMDFLLFCNYYRNDV VAGEALVRKLRFSMTDDEKEGIYADEAAKLWGKFRNDFENIADHMNGDVIKELGKADMDFDEKILDSEKKNASDLLYFSKMIYM LTYFLDGKEINDLLTTLI SKFDNIKEFLKIMKSSAVDVECELTAGYKLFNDSQRITNELFIVKNIASMRKPAASAKLTMFRDAL TILGIDDNITDDRISEILKLKEKGKGIHGLRNFITNNVIESSRFVYLIKYANAQKIREVAKNEKWMFVLGGIPDTQIERYYKS CVEFPDMNSSLEAKRSELARMIKNISFDDFKNVKQQAKGRENVAKERAKAVIGLYLTVMYLLVKNLWWARYVIAIHCLERDF GLYKEI IPELASKNLKNDYRILSQTLCELCDDRDESPNLFLKKNKRLRKCVEVDINNADSSMTRKYRNCIAHLTWRELKEYIG DIRTVDSYFSIYHYVMQRCITKRENDTKQEEKIKYEDDLLKNHGYTKDFVKALNSPFGYNIPRFKNLSIEQLFDRNEYLTEK
(SEQ ID NO: 349)
>OMWO01000091_3
[uncultured Clostridiales bacterium]
MAKKKRMSAKERKQQQINLRIKKATEDSTKKVNTTVAVNNKPISKEIKKSKAKLAGVKWVIKANDDVAYISSFGKGNNSVLEKR IIGDVSSDWKDSHMYWPKYTKKNYEIKNGFSSGSSLTTHPNKPDKNSGMDALCLKTYFEKEIFKDKFNDNMHIQAIYNIFDI EKTLAKHITNIIYAWSLDRSYIQSGNDTIGFGLNFNI PYAEYGGGKDSNGKPENKSAWEKRESFIKFYNNAKDRFGYFESVFY QNGKQI SEEKFYIYLNILNFVRNSTFHYNNTSSHLYKERYCKINPKNNLKTDFEFVSYLNEFVKNKFKNVNKNFISNEKNNLYI ILNAYGEDIEDVEWKKYSKELYKLSVLKTNKNLGVNVKKLRESAIEYGYCPLPYDKEKEVAKLSSIKHKLYKTYDFVITHYLN SNDKLLLEIVEALRLSKNDDKKENVYKIYAEKIFKAEYVINPIKTI SNLFAEKGDKLFNEKVSI SEEYVEDIRIDKNIHNFTKV IFFLTCFLDGKEINDLLTNIISKLQVIEDHNNVIKAIANNNDAVYKDYSDKYAVFKNSGKIATELEAIKSIARMENKINKAFKE PLLKDAMLALGVSPNDLDEKYEKYFKTDVDADKDHQKVSTFLMNNVINNSRFKYWKYINPADINRLAKNKHLVKFVLDQIPHK QIDSYYNSVSTVEEPSYKGKIQLLTKKITGLNFYSLFENCKI PNVEKEKKKAVITLYFTI IYILVKNLWINGLYTLALYFVER DGFFYKKICEKKDKKKTNKDVDYLLLPEIFSGSKYREETKNLKLPKEKDREIMKKYLPNDEDRKEYNKFFKQYRNNIVHLNIIA NLSKLTSTIDKEINSYFEIFHYCAQRVMFDYCKNNNKWL (SEQ ID NO: 350)
Table 3. Representative Type VI-D Direct Repeat Nucleotide Sequences
Figure imgf000139_0001
364)
CDZT01047721_3 (SEQ ID NO: 11) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 368)
CDZU01022944_3 (SEQ ID NO: 11) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 369)
CDZV01031905_3 (SEQ ID NO: 11) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 370)
OGPA01000243_2 (SEQ ID NO: 11) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 410)
3300007296 | Ga0104830_100502_31 (SEQ
ID NO: 12) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 38)
3300007296 | Ga0104830_100502_31 (SEQ
ID NO: 12) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 74)
CTACTACACTAGTGCGAATTTGCACTAGTCTAAAACTA (SEQ ID NO:
ODXP01000624_4 (SEQ ID NO: 12) 547)
3300007299 | Ga0104319_1000623_29 (SEQ
ID NO: 13) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
ODKA01005851_3 (SEQ ID NO: 13) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OGPQ01001037_3 (SEQ ID NO: 14) CTACTACACTGGTGCAAATTTGCACTA (SEQ ID NO: 414)
33000084961Ga0115078_100057_51 (SEQ
ID NO: 14) CTACTACACTGGTGCAAATTTGCACTA (SEQ ID NO: 557)
CDZK01015063_14 (SEQ ID NO: 15) TACTGGTGCGAATTTGCACTAA (SEQ ID NO: 365)
3300001598 |EMG 10000232 1 (SEQ ID NO:
16) GGACAATAACCTGCGAATTTTGGCAGGTTCTATGAC (SEQ ID NO: 36)
3300001598 |EMG 10003641 1 (SEQ ID NO:
17) GAACTACACCCCTGCAGAAATGCTGGGGTCTGAAAC (SEQ ID NO: 35)
3300018494 | Ga0187911_10005861_19
(SEQ ID NO: 18) GAACTACAGCCCTGTGAAATAACGGGGTTCTAAAAC (SEQ ID NO: 46)
3300018494 | Ga0187911_10005861_19
(SEQ ID NO: 18) GAACTACAGCCCTGTGAAATAACAGGGTTCTAAAAC (SEQ ID NO: 47)
3300018494 | Ga0187911_10005861_19
(SEQ ID NO: 18) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 562)
3300018495 | Ga0187908_10006038_18
(SEQ ID NO: 18) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 565)
3300018475 | Ga0187907_10006632_17
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGATAGGGGGTTGAAAC (SEQ ID NO: 44)
3300018494 | Ga0187911_10005861_18
(SEQ ID NO: 19) GAACTACAGCCCTGTGAAATAACGGGGTTCTAAAAC (SEQ ID NO: 46)
3300018494 | Ga0187911_10005861_18
(SEQ ID NO: 19) GAACTACAGCCCTGTGAAATAACAGGGTTCTAAAAC (SEQ ID NO: 47)
3300018475 | Ga0187907_10006632_17
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 558)
3300018475 | Ga0187907_10006632_17
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 559)
3300018493 | Ga0187909_10005433_18
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 560)
3300018493 | Ga0187909_10005433_18
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 561)
3300018494 | Ga0187911_10005861_18
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 563)
33000184951Ga0187908_10006038_19
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 566)
33000188781Ga0187910_10006931_17
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 567)
33000188781Ga0187910_10006931_17
(SEQ ID NO: 19) CATGTAAACCCCTAACAAATGGTAGGGGTTTGAAAC (SEQ ID NO: 568)
33000184941Ga0187911_10069260_3 (SEQ
ID NO: 20) GAACTACAGCCCTGTGAAATAACAGGG (SEQ ID NO: 564)
3300018493 | Ga0187909_10030832_9 (SEQ
ID NO: 21) CTACTACTACCCTGTTATTTGACAGGGTTCAAAAAC (SEQ ID NO: 45)
33000184941Ga0187911_10019634_9 (SEQ CTACTACTACCCTGTTATTTGACAGGGTTCAAAAAC (SEQ ID NO: 45) ID NO: 21)
3300018878 | Ga0187910_10040531_l (SEQ
ID NO: 21) GTTTCTGAACCCTGCCATTTGGCAGGGTAGTAGTTG (SEQ ID NO: 569)
3300018493 | Ga0187909_10024847_5 (SEQ
ID NO: 22) GAACGACGTCACTACACACCGAGAGGTGTCTAAAAC (SEQ ID NO: 48)
3300018494 | Ga0187911_10037073_4 (SEQ
ID NO: 22) GAACGACGTCACTACACACCGAGAGGTGTCTAAAAC (SEQ ID NO: 48)
3300018495 | Ga0187908_10013323_2 (SEQ
ID NO: 22) GAACGACGTCACTACACACCGAGAGGTGTCTAAAAC (SEQ ID NO: 48)
3300018878 | Ga0187910_10015336_15
(SEQ I D NO: 22) GAACGACGTCACTACACACCGAGAGGTGTCTAAAAC (SEQ ID NO: 48)
3300018878 | Ga0187910_10015336_15
(SEQ I D NO: 22) CAACTACTACCCTGCCAAATGGCAGGGTTCAGAAAC (SEQ ID NO: 49)
WP_074833651.1 (SEQ ID NO: 23) CCCTTTGTACTATACCTGTTTTACACAGGTCTAAAAC (SEQ ID NO: 60)
WP_074833651.1 (SEQ ID NO: 23) GTACTATACCTGTTTTACACAGGATAATAACCAAAAT (SEQ ID NO: 61)
WP_074833651.1 (SEQ ID NO: 23) CTACTATACTAGTGTGATTTTACACTAGTCTAAAAC (SEQ ID NO: 352)
WP_041337480.1 (SEQ ID NO: 24) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 63)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
WP_041337480.1 (SEQ ID NO: 24) (SEQ ID NO: 353)
DBYI01000091_43 (SEQ ID NO: 25) GAACTATACCCCTACCAAATGGTCGGGGTCTGAAAC (SEQ ID NO 64)
WP_075424065.1 (SEQ ID NO: 26) CAAGTAAACCCCTACCAACTGGTCGGGGTTTGAAAC (SEQ ID NO 65)
WP_075424065.1 (SEQ ID NO: 26) CAAGTAAACCCTTACCAACTGGTCGGGGTTTGAAAC (SEQ ID NO 66)
WP_009985792.1 (SEQ ID NO: 27) GAACTATAGTAGTGTAAATTTGCACTACTATAAAAC (SEQ ID NO 67)
WP_009985792.1 (SEQ ID NO: 27) GAACTATAGTAGTGTGAATTTACACTACTCTAAAAC (SEQ ID NO 354)
CDC65743.1 (SEQ I D NO: 28) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 68)
CDC65743.1 (SEQ I D NO: 28) CTACTACACTAGTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 69)
CDC65743.1 (SEQ I D NO: 28) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 70)
CDC65743.1 (SEQ I D NO: 28) CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO 71)
CDC65743.1 (SEQ I D NO: 28) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO: 356)
DJXD01000002_3 (SEQ ID NO: 29) CAACTACAACCCCGTAAAAATACGGGGTTCTGAAAC (SEQ ID NO: 73)
DJXD01000002_3 (SEQ ID NO: 29) CAACTACAACCCCGTAAAAATACGGGGTTCTGAAACC (SEQ ID NO: 357)
SCH71549.1 (SEQ ID NO: 30) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAT (SEQ ID NO 57)
SCH71549.1 (SEQ ID NO: 30) CTACTACACTAGTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 58)
SCH71549.1 (SEQ ID NO: 30) CTACTACACTAGTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 69)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAA (SEQ ID NO: 367)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 409)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 415)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 488)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 514)
SCH71549.1 (SEQ ID NO: 30) GTGCGAATTTGCGCTAGTCTAAAAC (SEQ ID NO 526)
SCJ27598.1 (SEQ I D NO: 31) CTACTACACTGGTGCAAATTAGCACTAGTCTAAAAC (SEQ ID NO: 76)
SCJ27598.1 (SEQ I D NO: 31) CTACTACACTGGTGCAAATTAGCACTAGTCTAAAAC (SEQ ID NO: 77)
SCJ27598.1 (SEQ I D NO: 31) CTACTACACTGGTGTGAATTTGCAC (SEQ ID NO: 487)
NZ_ACOK01000100_5 (SEQ ID NO: 200) GAACTATAGTAGTGTAAATTTGCACTACTATAAAAC (SEQ ID NO: 67)
NZ_ACOK01000100_5 (SEQ ID NO: 200) GAACTATAGTAGTGTGAATTTACACTACTCTAAAAC (SEQ ID NO: 355)
3300006226 | Ga0099364_10024192_5 (SEQ
ID NO: 201) GTGCAGTAGCCTTACAGATTCGTAGGGTTCTGAGAC (SEQ ID NO: 37)
3300007361 | Ga0104787_100954_14 (SEQ
ID NO: 202) CTACTACACAGGTGCAATTTTGCACTAGTCTAAAAC (SEQ ID NO: 40)
3300007361 | Ga0104787_100954_14 (SEQ
ID NO: 202) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 41)
CDZK01015063_14 (SEQ I D NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO 366)
OIZB01000622_13 (SEQ ID NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO 498)
OIZB01000622_13 (SEQ ID NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO 499)
ODHZ01001211_7 (SEQ I D NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO 537)
3300007361 | Ga0104787_100954_14 (SEQ TACTGGTGCGAATTTGCACTAA (SEQ ID NO 554) ID NO: 202)
33000073611 Ga0104787_100954_14 (SEQ
ID NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO: 555)
33000082721 Ga0111092_1001379_l (SEQ
ID NO: 202) TACTGGTGCGAATTTGCACTAA (SEQ ID NO: 556)
CEAA01017658_2 (SEQ ID NO: 203) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OCHE01000387_10 (SEQ ID NO: 203) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 392)
OCTW011587266_5 (SEQ ID NO: 204) CTTATACAACACCCATTTTCACAGTGGGT (SEQ ID NO: 371)
OCVV011003687_3 (SEQ ID NO: 205) GTTTGAGAGTAGTGTAATTTTATAGGGTAGTAAAAC (SEQ ID NO: 372)
OCVV011003687_3 (SEQ ID NO: 206) GTTTGAGAGTAGTGTAATTTTATAGGGTAGTAAAAC (SEQ ID NO: 373)
ODAI010069496_4 (SEQ ID NO: 207) GAACTATAGTAGTGTTTTTTTACACT (SEQ ID NO: 374)
ODAI011611274_2 (SEQ ID NO: 208) GTACTACACCCCTGCAGTTTTGCAGGGGTCTGAAAC (SEQ ID NO 375)
OATA01000148_47 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 376)
OBAI01000753_39 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 379)
OBAQ01000162_41 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 380)
OCHU01001749_1 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 393)
OCPV01000148_47 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 396)
OFMN01000509_2 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 397)
OFRY01000077_43 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 400)
OGRH01000378_2 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 427)
OGUL01000592_19 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 432)
OIGD01000177_59 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 492)
OIXV01006344_7 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 495)
PPYF01129432_15 (SEQ ID NO: 209) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 531)
OAVJ01001264_7 (SEQ ID NO: 210) CTACTACACTGGTGCAAATTTGCACTA (SEQ ID NO: 377)
OBAE01000973_3 (SEQ ID NO: 211) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 378)
OBAR01000289_55 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OBAS01000138_55 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OCHD01001741_1 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OCHK01000325_37 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OCHS01000450_6 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OCQA01000142_55 (SEQ ID NO: 212) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OBCV01000332_2 (SEQ ID NO: 213) CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO 75)
OBDE01000870_1 (SEQ ID NO: 214) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO: 381)
OBII01002626_5 (SEQ ID NO: 215) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OBII01002626_3 (SEQ ID NO: 216) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OBJF01000033_8 (SEQ ID NO: 217) GATTGAAAGGATTGTAAATTTGCAAGGTCTTAAAAC (SEQ ID NO 382)
OBJF01000033_8 (SEQ ID NO: 218) GATTGAAAGGATTGTAAATTTGCAAGGTCTTAAAAC (SEQ ID NO 383)
OJMK01000275_31 (SEQ ID NO: 218) GATTGAAAGGATTGTAAATTTGCAAGGTCTTAAAAC (SEQ ID NO 508)
OBKG01000025_26 (SEQ ID NO: 219) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OBKR01000858_3 (SEQ ID NO: 220) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 384)
OJMI01000733_4 (SEQ ID NO: 220) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 507)
OBVH01003037_1 (SEQ ID NO: 221) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAAC (SEQ ID NO: 385)
OBVH01003037_2 (SEQ ID NO: 222) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAAC (SEQ ID NO: 386)
OBVY01000267_8 (SEQ ID NO: 223) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 387)
OGOC01002653_3 (SEQ ID NO: 223) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 403)
OBXZ01000094_20 (SEQ ID NO: 224) GATTGAATGGATTGTAAATTT (SEQ ID NO: 388)
OBXZ01000094_20 (SEQ ID NO: 225) GATTGAATGGATTGTAAATTT (SEQ ID NO: 389)
OCHB01002119_1 (SEQ ID NO: 226) ACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO: 390)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
OCHC01000012_250 (SEQ ID NO: 227) (SEQ ID NO: 391)
OCPS01000464_4 (SEQ ID NO: 227) GCTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 394)
OCHN01000290_35 (SEQ ID NO: 228) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OGPS01000672_3 (SEQ ID NO: 229) CTACTACACTAGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO 39)
OCPQ01000020_138 (SEQ ID NO: 229) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OCPU01001206_17 (SEQ ID NO: 230) GCTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 395) OEHT01000244_15 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OGPU01000173_30 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHHR01000227_3 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OJOL01000697_12 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OFMU01000310_31 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO: 398)
OGOI01001249_5 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO: 404)
OGQV01000794_21 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACT (SEQ ID NO: 419)
OGQZ01000194_33 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACT (SEQ ID NO: 422)
OHPC01000165_40 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 473)
OHUN01000170_40 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 486)
OJNT01000812_6 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 512)
OJOF01000269_30 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 513)
OKSV01000264_32 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 520)
OKVF01000105_32 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 525)
OEBA01002798_7 (SEQ ID NO: 231) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO 550)
OFMV01000268_25 (SEQ ID NO: 232) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 399)
OGQU01002289_9 (SEQ ID NO: 232) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 418)
OLGH01000826_1 (SEQ ID NO: 232) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 527)
ODVS01001471_9 (SEQ ID NO: 232) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 543)
OGCM01002738_3 (SEQ ID NO: 233) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO: 401)
OGCO01000353_15 (SEQ ID NO: 234) ACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO: 402)
OGOK01000323_15 (SEQ ID NO: 235) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 405)
OGOL01000786_27 (SEQ ID NO: 236) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 406)
OGOO01001137_18 (SEQ ID NO: 237) GAATTTGCACTAGTCTAAAAC (SEQ ID NO: 407)
GGAGGTGATAAAAATGGGAAAGACGATCCTTACGGCTATC (SEQ ID NO:
OGOP01001824_10 (SEQ ID NO: 238) 408)
GGAGGTGATAAAAATGGGAAAGACGATCCTTACGGCTATC (SEQ ID NO:
OGRT01000617_3 (SEQ ID NO: 238) 430)
OGPB01000314_7 (SEQ ID NO: 239) CTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 411)
OGPJ01000449_26 (SEQ ID NO: 240) CTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 412)
OGPK01001709_2 (SEQ ID NO: 240) CTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 413)
OGPS01000624_23 (SEQ ID NO: 241) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 34)
OGQH01000331_48 (SEQ ID NO: 242) CCTACTACACTGGTGCGAATTTGCACTA (SEQ ID NO: 416)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
OGQX01000605_8 (SEQ ID NO: 242) (SEQ ID NO: 421)
OGRG01000028_3 (SEQ ID NO: 242) GCTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 426)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
ODEE01001565_1 (SEQ ID NO: 242) (SEQ ID NO: 532)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
ODIH01000145_73 (SEQ ID NO: 242) NO: 538)
OGQO01007270_2 (SEQ ID NO: 243) CTACTACACTGGTGCGAATTTGCACTA (SEQ ID NO: 417)
OEFH01000394_40 (SEQ ID NO: 243) CTACTACACTGGTGCGAATTTGCACTA (SEQ ID NO: 552)
OGQW01001429_6 (SEQ ID NO: 244) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO: 420)
OGRA01000610_24 (SEQ ID NO: 245) ACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 423)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
OGRE01001635_6 (SEQ ID NO: 246) NO: 424)
OGRF01000967_2 (SEQ ID NO: 247) GATTTTGCACTAGTCTAAAAC (SEQ ID NO: 425)
OGRN01001989_2 (SEQ ID NO: 248) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 428)
OGRQ01003333_5 (SEQ ID NO: 249) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 429)
OGRU01000829_2 (SEQ ID NO: 250) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 431)
OGSD01001176_18 (SEQ ID NO: 251) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 42)
OGWY01002732_3 (SEQ ID NO: 252) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 34)
OGXI01000433_6 (SEQ ID NO: 253) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 433)
OGYU01002161_4 (SEQ ID NO: 253) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 436)
OGGS01001705_3 (SEQ ID NO: 253) GGAGGTGATAAAAATGGGAAA (SEQ ID NO 574)
OGXJ01002463_5 (SEQ ID NO: 254) CTACTACACTGGTGCGAATTTG (SEQ ID NO: 434)
OGXL01002096_10 (SEQ ID NO: 255) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 42) OGYD01000683_23 (SEQ ID NO: 256) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 435)
OGYL01002810_3 (SEQ ID NO: 257) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OGYY01000371_37 (SEQ ID NO: 258) TTTGCACTAGTCTAAAAC (SEQ ID NO: 437)
OHBM01000552_13 (SEQ ID NO: 258) TTTTGCACTAGTCTAAAACTT (SEQ ID NO: 443)
OGGV01005531_2 (SEQ ID NO: 258) TTTTGCACTAGTCTAAAACTT (SEQ ID NO: 575)
OGZC01000639_10 (SEQ ID NO: 259) GTTTTAGTATCCACGATAAACGTGGATTGTAGT (SEQ ID NO: 438)
OHAI01000724_7 (SEQ ID NO: 260) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHAJ01000052_20 (SEQ ID NO: 261) GATTGAAAGCTATGCGAATTTGCACAGTCTTAAAAC (SEQ ID NO 439)
OGDS01000069_10 (SEQ ID NO: 261) GATTGAAAGCTATGCGAATTTGCACAGTCTTAAAAC (SEQ ID NO 572)
OHAN01001071_11 (SEQ ID NO: 262) CTACTACACTAGTGCAAATTTGCGCTAGTCTAAAACT (SEQ ID NO: 440)
OHAR01000226_9 (SEQ ID NO: 263) CTACTACACTAGTGCGAATTTGCACTA (SEQ ID NO 441)
OHGN01001355_3 (SEQ ID NO: 263) CTACTACACTAGTGCGAATTTGCACTA (SEQ ID NO 454)
OHHD01000480_3 (SEQ ID NO: 263) CTACTACACTAGTGCGAATTTGCACTA (SEQ ID NO 456)
OHKC01000402_5 (SEQ ID NO: 263) CTACTACACTAGTGCGAATTTGCACTA (SEQ ID NO 460)
OHBL01000590_7 (SEQ ID NO: 264) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAA (SEQ ID NO: 442)
OHLO01000586_3 (SEQ ID NO: 264) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 463)
OHSZ01000559_4 (SEQ ID NO: 264) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 482)
OHBP01000023_129 (SEQ ID NO: 265) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHDS01000019_133 (SEQ ID NO: 265) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHMH01000024_3 (SEQ ID NO: 265) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHBQ01000429_2 (SEQ ID NO: 266) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHEL01001488_6 (SEQ ID NO: 266) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHKH01000861_3 (SEQ ID NO: 266) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHBW01001448_1 (SEQ ID NO: 267) ACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 444)
OHEG01001211_2 (SEQ ID NO: 267) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 451)
OHSG01000119_6 (SEQ ID NO: 267) CTACTATACTGGTGCGATTTTGCACTA (SEQ ID NO: 479)
OHSQ01001407_1 (SEQ ID NO: 267) ACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 481)
OHJG01000198_33 (SEQ ID NO: 268) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAT (SEQ ID NO: 59)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
OHCE01000125_17 (SEQ ID NO: 268) NO: 445)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
OHJJ01000127_35 (SEQ ID NO: 268) NO: 458)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
OHRD01000126_17 (SEQ ID NO: 268) (SEQ ID NO: 477)
OHCH01000211_3 (SEQ ID NO: 269) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 74)
OHPE01000834_1 (SEQ ID NO: 269) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 74)
OHFX01001477_3 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 453)
OHIJ01000315_7 (SEQ ID NO: 269) CTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 457)
OHMQ01000465_4 (SEQ ID NO: 269) CTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 467)
OHMW01000451_18 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 468)
OHNF01001864_4 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 469)
OHQE01002584_3 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 476)
OKSK01000361J.7 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 519)
OKTU01000352_17 (SEQ ID NO: 269) CTACACTGGTGCGAGTTTGCACTAGTCTAAAAC (SEQ ID NO 523)
OHCP01000044_27 (SEQ ID NO: 270) GTACTAAAGCCCGCTAGTATAGACGGGTTCTAAGAC (SEQ ID NO 446)
OHSM01000196_10 (SEQ ID NO: 270) GTACTAAAGCCCGCTAGTATAGACGGGTTCTAAGAC (SEQ ID NO 480)
OKTR01000164_10 (SEQ ID NO: 270) GTACTAAAGCCCGCTAGTATAGACGGGTTCTAAGAC (SEQ ID NO 522)
OHCW01000317_3 (SEQ ID NO: 271) GGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 447)
OHDC01002972_3 (SEQ ID NO: 271) GGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 448)
OHKW01000215_41 (SEQ ID NO: 271) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 461)
OHPP01000240_36 (SEQ ID NO: 271) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 475)
OHRM01001189_3 (SEQ ID NO: 271) GGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 478)
OHTG01000221_40 (SEQ ID NO: 271) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 483)
OHTH01000201_42 (SEQ ID NO: 271) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 484)
OKTJ01001834_4 (SEQ ID NO: 271) GGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 521)
QDFV01004017_1 (SEQ ID NO: 271) GGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 533) OHDP01000241_4 (SEQ ID NO: 272) TGAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 449)
OHFV01000201_5 (SEQ ID NO: 272) TGAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 452)
OHLY01001101_3 (SEQ ID NO: 272) TGAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 464)
OHPD01001131_4 (SEQ ID NO: 272) TGAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 474)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
OHDT01000502_2 (SEQ ID NO: 273) NO: 450)
OHFA01000290_5 (SEQ ID NO: 274) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OHJZ01000157_5 (SEQ ID NO: 274) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OHST01000977_4 (SEQ ID NO: 274) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OKSP01001453_2 (SEQ ID NO: 274) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OHGX01000264_3 (SEQ ID NO: 275) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 455)
OHME01000303_3 (SEQ ID NO: 275) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 465)
OHNP01000278_34 (SEQ ID NO: 275) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 470)
OHOI01000307_2 (SEQ ID NO: 275) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO 471)
OHIB01002708_3 (SEQ ID NO: 276) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OHJK01001285_9 (SEQ ID NO: 277) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OHSI01000544_10 (SEQ ID NO: 277) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OKSN01001169_3 (SEQ ID NO: 277) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OHJS01001864_3 (SEQ ID NO: 278) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 459)
OHLH01003112_3 (SEQ ID NO: 278) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAA (SEQ ID NO: 462)
OHJT01001977_4 (SEQ ID NO: 279) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 34)
OHPW01002065_2 (SEQ ID NO: 279) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 34)
OHMF01000395_24 (SEQ ID NO: 280) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACT (SEQ ID NO: 466)
OHOK01001322_2 (SEQ ID NO: 280) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 472)
OHUA01000395_26 (SEQ ID NO: 280) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 485)
OHUY01000263_2 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHVU01001109_1 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHXZ01000057_25 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OHYU01000376_4 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OICI01000194_18 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OIDC01000397_3 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OIDU01000174_25 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OKUL01000400_17 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OKUR01000327_17 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OKVB01000375_17 (SEQ ID NO: 281) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OKVC01000355_17 (SEQ ID NO: 281) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 524)
OIBN01003740_1 (SEQ ID NO: 282) CTACTACACTGGTGCAAATTAGCACTAGTCTAAAAC (SEQ ID NO: 77)
OIEE01000042_11 (SEQ ID NO: 283) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 489)
OIEL01000292_3 (SEQ ID NO: 284) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO: 490)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
OJMG01000332_24 (SEQ ID NO: 284) NO: 506)
OIEN01002196_3 (SEQ ID NO: 285) GCCCCTTGACCTTACGAAATGGTAAGGTTCCAAAAC (SEQ ID NO: 491)
OIXA01002812_3 (SEQ ID NO: 286) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OIXU01000818_5 (SEQ ID NO: 287) GATTGAAAGGATTGTAAATTT (SEQ ID NO: 493)
OIXU01000818_6 (SEQ ID NO: 288) GATTGAAAGGATTGTAAATTT (SEQ ID NO: 494)
OIYU01000175_4 (SEQ ID NO: 289) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO: 496)
OIZA01000315_9 (SEQ ID NO: 290) GATTGAAAGGTTTGTAAATTTACAAGGTCTTAAAAC (SEQ ID NO: 497)
OIZI01000180_12 (SEQ ID NO: 291) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAACA (SEQ ID NO: 500)
OIZI01000180_12 (SEQ ID NO: 292) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAACA (SEQ ID NO: 501)
GAAAGAAAACAAAAAGACGAGAACAGGACAAATGGCTTTCTGAGCAGGCT
OIZU01000200_48 (SEQ ID NO: 293) (SEQ ID NO: 502)
OIZW01000344_20 (SEQ ID NO: 294) GCTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 503)
OIZX01000427_25 (SEQ ID NO: 295) ACTATAGCCCTGCCGGAAA (SEQ ID NO: 504)
OIZX01000427_26 (SEQ ID NO: 296) ACTATAGCCCTGCCGGAAA (SEQ ID NO: 505)
OJMJ01002228_5 (SEQ ID NO: 297) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 74)
OJMM01002900_7 (SEQ ID NO: 298) GTACAATAGCCCTCTCGTAGTTGAGGGCTCTGAGAC (SEQ ID NO: 509) OJMM01002900_7 (SEQ ID NO: 299) GTACAATAGCCCTCTCGTAGTTGAGGGCTCTGAGAC (SEQ ID NO 510)
OJMN01000417_22 (SEQ ID NO: 300) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 42)
OJNI01000536_4 (SEQ ID NO: 300) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 42)
OJNR01001167_9 (SEQ ID NO: 301) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OJOP01001093_3 (SEQ ID NO: 301) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO 34)
OJNS01001527_9 (SEQ ID NO: 301) GAACTACACCCGTGCAAAATTGCAGG (SEQ ID NO: 511)
OJPG01000139_73 (SEQ ID NO: 302) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OJPS01000131_3 (SEQ ID NO: 302) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OJQH01000635_3 (SEQ ID NO: 302) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OJRP01000045_31 (SEQ ID NO: 302) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OJPX01000614_4 (SEQ ID NO: 303) GTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 515)
OJRG01001951_4 (SEQ ID NO: 303) GTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO: 516)
OGNV01000836_4 (SEQ ID NO: 304) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OKRZ01002949_5 (SEQ ID NO: 304) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
OKSB01002689_10 (SEQ ID NO: 305) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OKSC01004083_2 (SEQ ID NO: 306) GCACTACACCCCCCTGAAACATGAG (SEQ ID NO: 517)
OKSD01002505_11 (SEQ ID NO: 307) CTACTACACTAGTGCGAATTTGCACTA (SEQ ID NO: 518)
GAAAGAAAACAAAAAGACGAGAACAGGACAAATGGCTTTCTGAGCAGGCT
OLGN01000304_32 (SEQ ID NO: 308) (SEQ ID NO: 528)
OLHE01000257_41 (SEQ ID NO: 309) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
PPYE01106492_34 (SEQ ID NO: 310) GACGGGAGGTGATGAAAATG (SEQ ID NO: 529)
PPYE01385196_3 (SEQ ID NO: 311) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
PPYE01512733_3 (SEQ ID NO: 312) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO: 530)
PPYF01670242_39 (SEQ ID NO: 313) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
ODFW01000112_43 (SEQ ID NO: 314) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 534)
ODTU01003882_3 (SEQ ID NO: 314) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 541)
ODGN01000188_50 (SEQ ID NO: 315) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
ODHH01000275_14 (SEQ ID NO: 316) NO: 535)
GCTGAAAGAAAACAGAAAGACGAGGAGCAGGACAAATGGCTTTC (SEQ ID
ODYJ01000298_33 (SEQ ID NO: 316) NO: 549)
ODHP01001712_3 (SEQ ID NO: 317) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
ODHV01000466_16 (SEQ ID NO: 318) CTAGTGCAAATTTGCACTAGTCTAAAACG (SEQ ID NO: 536)
ODXE01000717_15 (SEQ ID NO: 318) CTAGTGCAAATTTGCACTAGTCTAAAACG (SEQ ID NO: 545)
ODJZ01000182_13 (SEQ ID NO: 319) CTACTACACTGGTGCGAATTTGCACTA (SEQ ID NO: 539)
ODLN01002572_7 (SEQ ID NO: 320) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 62)
ODQJ01000729_25 (SEQ ID NO: 321) CTACTATACTGGTGCGATTTTGCACTAGTCTAAAAC (SEQ ID NO 540)
ODUN01000242_23 (SEQ ID NO: 322) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 42)
ODWX01000843_3 (SEQ ID NO: 322) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 42)
CCTACTACACTAGTGCGAATTTGCACTAGTCTAAAACT (SEQ ID NO:
ODVQ01003982_3 (SEQ ID NO: 323) 542)
ODVR01002077_3 (SEQ ID NO: 324) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
ODXC01000747_3 (SEQ ID NO: 325) CTACTACACTGGTGCGAATTTGCACTA (SEQ ID NO: 544)
TCTCTTGGCGGAAAGAAAACAGAAAGACGAAGAACAGGACAAATGGCTATC
OEEK01000163_43 (SEQ ID NO: 325) (SEQ ID NO: 551)
ODXO01005124_2 (SEQ ID NO: 326) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 546)
OEFW01000634_7 (SEQ ID NO: 326) GTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 553)
ODYC01000377_16 (SEQ ID NO: 327) GGAGGTGATAAAAATGGGAAA (SEQ ID NO: 548)
OEJW01000623_11 (SEQ ID NO: 328) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
3300019376 | Ga0187899_10021543_4 (SEQ
ID NO: 329) TGAACGATAGCCTGCTGAAATATGCAGGTTCTAAGAC (SEQ ID NO: 570)
OGCZ01001955_1 (SEQ ID NO: 330) CTACTATACTGGTGCGAATTTGCACTAGTCTAAAATG (SEQ ID NO: 571)
OGDY01002059_17 (SEQ ID NO: 331) GAACTACACCCGTGCAAAAATGCAGGGGTCTAAAAC (SEQ ID NO: 43)
OGEU01000713_24 (SEQ ID NO: 332) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OGFM01002125_3 (SEQ ID NO: 333) GACAGGAGGTGATAAAAATG (SEQ ID NO: 573)
OGHW01002048_1 (SEQ ID NO: 334) CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO: 75)
QGIE01002059_21 (SEQ ID NO: 335) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAACC (SEQ ID NO: 576) OGII01000819_21 (SEQ ID NO: 335) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAACC (SEQ ID NO: 577)
CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACTCA (SEQ ID NO:
OGJI01000038_151 (SEQ ID NO: 336) 578)
CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACTCA (SEQ ID NO:
OGKE01000029_151 (SEQ ID NO: 336) 581)
CTACTACACTGGTGCGAATTTGCACTAGTCTAAAACTCA (SEQ ID NO:
OGKG01000020_152 (SEQ ID NO: 336) 582)
OGJK01007642_2 (SEQ ID NO: 337) GTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO: 579)
OGJY01000516_18 (SEQ ID NO: 338) CTACTACACTGGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO: 62)
OGKA01000617_2 (SEQ ID NO: 339) CTACTACACTGGTGCGAATTTGCACTAG (SEQ ID NO: 580)
OGKG01002483_14 (SEQ ID NO: 340) GAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 34)
OGKW01000585_4 (SEQ ID NO: 341) ACTGGTGCGAATTTGCACTGGTCTAAAAC (SEQ ID NO: 583)
OGU01000192_54 (SEQ ID NO: 342) CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO: 75)
OGLM01001314_21 (SEQ ID NO: 343) TGAACTACACCCGTGCAAAATTGCAGGGGTCTAAAAC (SEQ ID NO: 584)
OGMO01000062_69 (SEQ ID NO: 344) CTACTACACTGGTGCAAATTTGCACTAGTCTAAAAC (SEQ ID NO 75)
OGMP01001167_15 (SEQ ID NO: 345) CTACTACACTAGTGCGAATTTGCACTAGTCTAAAAC (SEQ ID NO 74)
OGUJ01000114_43 (SEQ ID NO: 346) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAAC (SEQ ID NO 585)
OGUJ01000114_45 (SEQ ID NO: 347) GATTGAAAGGATTGTAAATTTACAAGGTCTTAAAAC (SEQ ID NO 586)
OJKY01000879_3 (SEQ ID NO: 348) GTACTAAAGCCCGCTAGTATAGACGGGTTCTAAGAC (SEQ ID NO 587)
OUF01000187_58 (SEQ ID NO: 349) CTACTACACTGGTGCGATTTTGCACTAGTCTAAAACT (SEQ ID NO: 588)
OMWO01000091_3 (SEQ ID NO: 350) GATTGAAAGCTATGCGAATTTGCACAGTCTTAAAAC (SEQ ID NO: 589)
Table 4. Amino Acid Sequences of CaslSd Accessory Proteins WYL1
>SCH71532.1
[ Ruminococcus sp . CAG:57]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNTVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 78)
>SCJ27525.1
[human gut metagenorae]
MLILPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEINDGLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 79)
>WP_041337479.1
[Ruminococcus bicirculans]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 80)
>LARF01000048_7
[Ruminococcus sp . N15.MGS-57]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 81)
>CDYU01004315_3
[gut metagenome]
MSMTPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDGLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEIIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NTVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 82)
>CDYX01024884_5
[gut metagenorae]
MFIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKTDDNYKYIGIP LLNAFIKWQIEEIDDGLDDKSKEI IKSYLI SKFSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNIIKINEVIKQNGNRYYSFIYAYSNMYSREKRRIRLIPYRIISDEYKMYNYLVCLSDEKSAGKEFK ADSCRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 83)
>OGPQ01001037_4
[human gut metagenorae]
MSMTPSTFLPKRDTNIPYIAEVQSIPLSPSAYAVIVKDKSIFETSLFPNGGSVSMSSFLTRIFDSAYIASLKYKSEEYNGIPLL NAFVQWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYED YALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFKAD SYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 84)
>ODVQ01003982_4
[human metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLFPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 85)
>3300007299 I Ga0104319_1000623_28
[human-digestive system-homo sapiens]
MLIPPSTFLPKRKDGVPYIAEVQSIPLSPSAYAVIVKDKSIFETSLSPNSSVSMSSFLTRIFDSAYRASLKYKSEEYNGIPLLN AFVQWQIEEIDGSLDDKSKEIIRSYLISKLSAKYKKTKTENAVRVRLSICRDLYDTLSRVDLCYENKVYGSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNSNRYDNFIYAYSSMYSREKCRIRLI PYRIVSDEYKMYNYLVCLSDEKSVGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 86)
>CDYS01033339_20
[gut metagenome]
MGTENSSNEYQEARQHLSLSDAAWAVLQDDRQDFGGGRSWAGILNYVFAEYRDKADASISVAVERRRAQYEEKLVGVAAPAVRK AVLEALLADYTEELIKKAAQNGATPPDKESFKFRLDRDNYAFREQWLDSPDAQYYGGRFSRYLRAVLEEYAAKTVYQREAIYFD PQMRLIQASAANGELLRIRLKKGSEFEVRPYGVLGDRQETYHYLVGLSRPDGTREPEKASSFRLSNIVKLEVSFRRSGRLTEKE RTDIESSIRGKGVQFLVQQRETIRIRLTEDGRQNYGRQLHLRPAARERAEVDDGLYRWEYTFYCTEFQAKAYFLKFCGDAKWE PQSLRETFAQEYRSGLRACGEEP (SEQ ID NO: 87)
>CDTW01032418_59
[gut metagenome]
MGTENSSNEYQEARQHLSLSDAAWAVLQDDRRDFGGGRSWAGILNYVFTMYRDKADASVSVAVSRRREQLEEQLGGWSPAARD AVLDRLMEVYAGELAEKAMSDGAVAQQKEVFKFRLDRDNYAFREQWLDSPDAARYYGNRFSRYLRAVLEEYAAKTVYQREAIYF DPQMRLIRAAAANGELLRIRMKTGSSFEVRPYGVLGDRQETYHYLVGLSRPDGTRGPEKEFNFRLSKI IKLDVSFRRSGRLTEK ERTDIESSIRGKGVQFLAQQRETIRIRLTEEGRRDYGSQMHLRPPAQTRTAVDDGAYRWEYTFFCTEFQARAYFLKFCGEAKW EPQSLRDTLAQEYRSGLRACGEEP (SEQ ID NO: 88)
>OATA01000148_62
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIKEINDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EVYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 590)
>OAVJ01001264_6
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKNKSDDNYKYNGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 591)
>OBAE01000973_4
[human gut metagenorae]
MSMTPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 592)
>OBAQ01000162_28
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIRDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIKEINDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EVYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 593)
>OBAS01000138_57
[human gut metagenome]
MSMTPSTFLPKREGSVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMHSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKGAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 594)
>OBCV01000332_3
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPTAYSVIVRDKSIFETSLSPNGGSVSMSSFLTRIFDSAYRASLKYKSEEYNGIPLL NAFVQWQIEEIDGSLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSRDDLCYENKVYGSTLRRFLKAVYED YALLSDCERERLIFADNI IKINEVIKQNSNRYDNFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLICLSDEKSADKEFKAD SYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEDHVKSVKHLLNDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEM (SEQ ID NO: 595)
>OBKG01000025_25
[human gut metagenome]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 596)
>OBKR01000858_4
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSSYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIEDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYFSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 597)
>OBVY01000267_8
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSGDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKKI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 598)
>OCHC01000012_251
[human gut metagenome] MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 599)
>OCHE01000387_8
[human gut metagenorae]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 600)
>OCPQ01000020_137
[human gut metagenome]
MSMTPSTFLPKRDTNIPYIAEVQSIPLSPSAYAVIVKDKSIFETSLFPNGGSVSMSSFLTRIFDSAYIASLKYKSEEYNGIPLL NAFVQWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYED YALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFKAD SYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNTVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 601)
>OFMU01000310_30
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIKEINDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 602)
>OFMV01000268_23
[human gut metagenome]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYNGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 603)
>OGCO01000353_16
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPTAYSVIVRDKSIFETSLSPNGGSVSMSSFLTRIFDSAYRASLKYKSEEYNGIPLL NAFVQWQIEEIDGSLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSRDDLCYENKVYGSTLRRFLKAVYED YALLSDCERERLIFADNI IKINEVIKQNSNRYDNFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLICLSDEKSADKEFKAD SYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEDHVKSVKHLLNDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEI (SEQ ID NO: 604)
>OGOP01001824_8
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIRDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIKEINDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EVYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 605)
>OGPB01000314_5
[human gut metagenome]
MSMTPSTFLPKRDKNATYIAEVQSIPLSPSTYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRGLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFKEMRTLYVEGAEAYNREVEM (SEQ ID NO: 606)
>OGPJ01000449 25 [human gut metagenorae]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRKFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKDGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 607)
>OGPU01000173_31
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSGDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKKI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 608)
>OGPY01000296_5
[human gut metagenome]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNTVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 609)
>OGQH01000331_47
[human gut metagenome]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSASKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 610)
>OGQO01007270_l
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTRIFDSAYIASLKYKSEEYNGIPLLN AFVQWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NTVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 611)
>OGRA01000610_25
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEM (SEQ ID NO: 612)
>OGSD01001176_17
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSNDNYKYIGIP LLNAFVQWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKESHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 613)
>OGXI01000433_8
[human gut metagenome]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIRDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIKEINDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EVYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 614) >OGXJ01002463_4
[human gut metagenorae]
MSMTPSTFLPKREKNATYIAEVQSIPLSPAAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKTDDNYKYIGIP LLNAFIKWQIEEIDDGLDDKSKEI IKSYLI SKFSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNIIKINEVIKQNGNRYYSFIYAYSNMYSREKRRIRLIPYRIISDEYKMYNYLVCLSDEKSAGKEFK ADSCRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 615)
>OGXL01002096_9
[human gut metagenome]
MSMTPSTFLPKRDTNIPYIAEVQSIPLSPSAYAVIVKDKSIFETSLFPNGGSVSMSSFLTRIFDSAYIASLKYKSEEYNGIPLL NAFVQWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYED YALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFKAD SCRI SRLSGLSIAEKLSQKEYSCVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 616)
>OGYY01000371_36
[human gut metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDGLDDKSKEIIKSYLISKLSAKYKKTKTENAVRVRLSISRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS CRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 617)
>OHAI01000724_6
[human gut metagenome]
MLIPTSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKNKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRLIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 618)
>OHAN01001071_10
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEI IKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVKM (SEQ ID NO: 619)
>OHAR01000226_10
[human gut metagenome]
MLIPPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 620)
>OHBL01000590_6
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKS NAVNEFISPPIQVKYYFNKFGKDGVI ISPSDSFEEMRTLYVEGAEAYNRWEM (SEQ ID NO: 621)
>OHBW01001448_2
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKHEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRLIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 622)
>OHCE01000125_19
[human gut metagenorae]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRGLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLNDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 623)
>OHCW01000317_6
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSNDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 624)
>OHEL01001488_5
[human gut metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLFPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYTLLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYSREVEM (SEQ ID NO: 625)
>OHFX01001477_2
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NTVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 626)
>OHGX01000264_3
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKHEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 627)
>OHJS01001864_5
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNSSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDDIDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 628)
>OHKC01000402_6
[human gut metagenome]
MLIPPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGKAQ AQRCQRVHLPAYPSQILFQ (SEQ ID NO: 629)
>OHMF01000395_25
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIRDKSIFETSLSPNGNVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDDLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEM (SEQ ID NO: 630)
>OHUY01000263_5
[human gut metagenorae]
MLMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKKI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSEGKEFK ADSYRI SRLSGLSISEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 631)
>OIXA01002812_2
[human gut metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSTYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 632)
>OIYU01000175_5
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 633)
>OIZW01000344_21
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVQWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRLIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGI ILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 634)
>OJMJ01002228_2
[human gut metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCKRERLLFAENI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRI SLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGRVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 635)
>OJMN01000417_21
[human gut metagenome]
MSMTPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEINDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 636)
>OJOH01001697_5
[human gut metagenome]
MLIPPSTFLPKRDKNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNTVNEFI SPPIQVKYYFNRFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 637)
>OJPG01000139_77
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 638)
>OJPX01000614_6
[human gut metagenorae]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSRLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKDNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 639)
>OKRZ01002949_4
[human gut metagenome]
MFIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKGYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNDSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 640)
>OKSD01002505_10
[human gut metagenome]
MLIPPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNDIPLLN AFVKWQIEEIDDGLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLLFADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISQPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 641)
>OLGN01000304_31
[human gut metagenome]
MSMTPSTFLPKRDTNIPYIAEVQSIPLSPSAYAVIVKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 642)
>OLHE01000257_40
[human gut metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKTDDNYKYIGIP LLNAFVKWQIEEIGDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEM (SEQ ID NO: 643)
>PPYE01106492_32
[human gut metagenome]
MSMTPSTFLPKRDTNVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 644)
>PPYE01385196_4
[human gut metagenome]
MLIPPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSTNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDDLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSI SRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 645)
>PPYE01512733_2
[human gut metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NTVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 646)
>ODFW01000112_41
[human metagenorae]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKHEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 647)
>ODGN01000188_49
[human metagenome]
MSMTPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 648)
>ODHH01000275_15
[human metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDSLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSLTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEI IKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 649)
>ODHP01001712_4
[human metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRGLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 650)
>ODHV01000466_16
[human metagenome]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSTNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGLP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYKKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSLKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 651)
>ODHZ01001211_6
[human metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSTGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPNDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 652)
>ODJZ01000182_15
[human metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 653)
>ODVR01002077_4
[human metagenome] MLIPPSTFLPKRDKNATYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEK (SEQ ID NO: 654)
>ODXC01000747_4
[human metagenorae]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTRIFDSAYIASLKYKSEEYNGIPLLN AFVQWQIEEIDDSLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS YRISRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFISPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 655)
>ODXO01005124_l
[human metagenome]
MSMTPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIRDKSIFETSLSPNGNVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDDLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 656)
>ODYC01000377_17
[human metagenome]
MLIPPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDSLDDKSKEI IKSYLI SKLSAKHEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEI IKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 657)
>OEJW01000623_13
[human metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYNGIP LLNAFVEWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 658)
>OGHW01002048_2
[metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKDI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVKGAEAYNREVEM (SEQ ID NO: 659)
>OGIE01002059_22
[metagenome]
MSMTPSTFLPKREKNATYIAEVQSIPLSPAAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYNGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKHRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSRLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNER PKHNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNRWEK (SEQ ID NO: 660)
>OGJI01000038_150
[metagenome]
MSMIPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPYGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSCRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFDEMRTLYVEGAEAYNREVEM (SEQ ID NO: 661) >OGJY01000516_19
[metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVEWQIEEIDDGLDDKSKEI IKGYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLIFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTKYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 662)
>OGKA01000617_3
[metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRLI RLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFK ADSYRI SRLSGLSIAEKLSQKEYFSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFEEMRTLYVEGAEAYNREVEI (SEQ ID NO: 663)
>OGKE01000029_150
[metagenome]
MIPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPYGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIPLL NAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYED YALLSDCERERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRI RLIPYRIVSDEYKMYNYLVCLSDEKSAGKEFKAD SCRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEKPK PNAVNEFI SPPIQVKYYFNKFGKDGVILSPSDSFDEMRTLYVEGAEAYNREVEM (SEQ ID NO: 664)
>OGLJ01000192_55
[metagenome]
MLIPPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCKRERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRI RLIPYRIVSDEYKMYNYLVCLSDEKSSGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEVHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 665)
>OGMO01000062_68
[metagenome]
MSMTPSTFLPKREGGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSEKYNGIPLLN AFVKWQIEEIDDGLDDKSKEIIKSYLISKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVYEDY ALLSDCERERLI FADNIIKINEVIKQNGSRYYSFIYAYSNMYSREKRRIRLI PYRIVSDEYKMYNYLVCLSDEKSAGKEFKADS CRISRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDISKVYLTEKGVEMFGKILYQRPILKGNEKPKP NAVNEFI S PPIQVKYYFNKFGKDGVI LS PSDS FEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 666)
>OGMP01001167_14
[metagenome]
MSMAPSTFLPKREDGVPYIAEVQSIPLSPSAYSVIIKDKSIFETSLSPNGSVSMSSFLTSIFDSAYIASLKYKSDDNYKYIGIP LLNAFVKWQIEEIDDGLDDKSKEI IKSYLI SKLSAKYEKTKTENAVRVRLSICRDLYDTLSSDDLYYENKVYSSTLRRFLKAVY EDYALLSDCKRERLLFADNI IKINEVIKQNGSRYYSFIYAYSNMYSREKRRI RLIPYRIVSDEYKMYNYLVCLSDEKSSGKEFK ADSYRI SRLSGLSIAEKLSQKEYSSVTEYERLKEGHVKSVKHLLSDPRFGSDESDI SKVYLTEKGVEMFGKILYQRPILKGNEK PKPNAVNEFI SPPIQVKYYFNKFGKDGVII SPSDSFEEMRTLYVEGAEAYNREVEM (SEQ ID NO: 667)
Table 5. Amino Acid Sequences of CaslSd Accessory Proteins WYL-bl
>DBYI01000091_50
[ Ruminococcus flavefaciens ]
MENKGKQREFIKDYNKIVPFLEKVFYYGTFSSEDYEKMDMMKKSKYSDYKRILEFAFRDVLYEKKNINGKKALGLRIDHFYDPH RAFLRFFTLKSFVSIERLFLTCYILKRI SKKGKCTINDICIGLDEVSVDDEVKDRKSTISRI IKNMVDYGFLIKKGSAYSINTG AKTLNNVALLNLIDICTNAYPI SICGSCIQNKIDQNYQSPFLIKHLHLGQIFNDELIWKLLIYANEKKQLCIELKKGIKLRELL PYRI ITNRETGRQYLFAIYVGTNNFDEYLMLRLDKI SDIKIEASECEI PDDTVLKEKYDTAFRYSFNGTTFLKRDQQPESGILV YDKSFEWNIKKHFPYSDAVSVDEKHNKVSIKVNTLTELKPWLRRNYDKVSLVESSDDTVDKMCDELKKWRKMYGII (SEQ ID NO : 89)
>SFX39521.1
[Ruminococcus flavefaciens ] MANEEKNRSFFKITTYENFRRFLKTNFYYCSLSQGQQGMFIKSIGTTKYNEYKNIIELIAGGKIEFPKINKRLAFRYNISQLES DYNELANSFQLRTLTSLDACLTLYILLFLSDKEMGSSDIYNRIGDIDFDIDEKTIRGKLKNMCEYGMI SYKNKKYSLNECSLYS VDTSIMLSLLNRADFMKNLVYPEVLGYDLFAALKKIYEERTGNEYI SPFQFKYSHLANILDDNVLWTLIEAIDNRQHVAFEYGG KIKERLIPVKIFTENEYNRCYLFAVKRFRNKLKFFVFRLSKIYNLKITNSDEDITEADFKEYSELYDSEKKCSFFGKIDSSAQN DTVELKYKRGIRSQLERDFSCIEFRKNYTAIVTVKSKKMMIPYLPANMGLIRTTDDELSGILNEDIEEMKKNYGII ( SEQ ID NO: 90)
>3300018494 I GaOl 87911_10005861_21
[mammals-digestive system- feces]
MNVI IKQGDI FMGNEERNRSFFKEDTYETFRKFLKTNFYYCTLSQKQQSEYVKYIGTTQYNHYRGI IERI SEGKISFKKYNKKK AFKYDVSQFASDYNVLANSFQLKTITASQTCLTIYILCVLAKSSLTRKGIVAAIADGIDEKTIVSRIKSMKEAGLI SYDGEKYF IEESIFYSMDESLLLRLLNMVDFMKNLVYPEALGYNLFDI IKKIYDDRLCVDYYSPFQLKYSHLANILDDNVLWSLIEAIEERQ YI SFIYKNEKKERI IPVKLFTENEYARRYLFAVKKFGNNYKKFI FRLSEIYNIKVMEKEVSVSKEEFGKLLEMYETESGYSFSG KIAPSSKTVSIKLRYKGRLKNQIERDFSNVKFEKGNTAEILIKNKKMIIPYLRSNMQLIQSTDEELSQKINSEIMEMKKLYGII
(SEQ ID NO: 91)
>DBYI01000091_49
[ Ruminococcus flavefaciens ]
MQSAWGILSLYGRYGI IIVIRGCDMENKGKQREFIKDYNKIVPFLEKVFYYGTFSSEDYEKMDMMKKSKYSDYKRILEFAFRDV LYEKKNINGKKALGLRIDHFYDPHRAFLRFFTLKSFVSIERLFLTCYILKRI SKKGKCTINDICIGLDEVSVDDEVKDRKSTIS RI IKNMVDYGFLIKKGSAYSINTGAKTLNNVALLNLIDICTNAYPI SICGSCIQNKIDQNYQSPFLIKHLHLGQIFNDELIWKL LIYANEKKQLCIELKKGIKLRELLPYRI ITNRETGRQYLFAIYVGTNNFDEYLMLRLDKI SDIKIEASECEI PDDTVLKEKYDT AFRYSFNGTTFLKRDQQPESGILVYDKSFEWNIKKHFPYSDAVSVDEKHNKVSIKVNTLTELKPWLRRNYDKVSLVESSDDTVD KMCDELKKWRKMYGII (SEQ ID NO: 668)
>3300018494 I GaOl 87911_10019634_8
[mammals-digestive system- feces]
MSADLGRNKLLLNENTLKIAKGAFYYGCFTVKHFEEQGISKSTYNRCKDFLLHVFQDRIEEINVPHSRTRMLRLKNDQFEDACN LLLDLFTYQPASSIEIVTFLSVLRVFTVAAPETSYTFENINKPI SHICEDRRTFKKKLHTLVDRGYLLCERRDKRSFQYRLAPV IFDRLDEFALYRLNALVDLCKCIYHPATCGRYLLDTLAFFNQQKSVNDETIFFCKHMHMGQVFDDAVLWKLMTAIYEKKI ISFT WGKSYRFQQPCRIIINESDGRRYLYSIGLNTYTKNGKMHRIDQISGIKEEKHTDEISVFSSEEADRRYHNSTQGSFNGISMPR KKRETAVLVYKKESYPEIQRHFPDAVPEVYDDDHDQVQITWSLKDIKPWLRLHLGEIRLQSTSNDVKDEFEKEMAEWRAMYGI
V (SEQ ID NO: 669)
Table 6. Amino Acid Sequences of CaslSd Accessory Proteins WYL-b2
>SFX39545.1
[Ruminococcus flavefaciens ]
MELFNEYRNKSLRAFLKLAERI SYGEELSIDEFEAEYYRLSGDNKKITSVFYKNTLYNDKLPIFDTREGKVRLFGEPDKCSNKH ISDTLLKSEITWLHNALNDKLSKLFLSDEERI SIDAKLSDYTEYYKNIDDMWRSNEDI SEEVEKNFKI ILKAINEKQALSYTFK NKNCEGFPVRIEYDERTCRIYMIIYDGNRFVKSDISKLSDIYITENSIDTIPEIKDDMLNKKAYLPWFTVTDDKNRKAIDRAL LAFSVYDHWEPIDEKTARFTIQYYTMDLDLLIKDILAFGSDIKVESPRYWKRITDILRKV (SEQ ID NO: 92)
>3300018494 I GaOl 87911_10005861_20
[mammals-digestive system- feces]
MELFNEFRNKSFNAFITLAERIANDNAVFSKTEFETEYYRLSGDENRITSIFYNNVINNEKYQI FTIPKDSKDKVQLSIEFDNK DDINIANI PITSEKKWLHSALHDKLSKLFLSDEEISYIDETI SEFPLYYEHIDDSWRKGENI SEESVINFRI ILQAINEKKSLS YKYNGKDSEGSPVKIEYDERTCKIYMILYNGSRFIKSDISGLSDICIKEQLYEKIPDIKEGMLEKKARHPIVFTVTDNKNRKSI ERALLAFSVYEHYVEPIDKNTAKFTIHYYTMDLDILIKDILAFGADIKVEAPQFWKKIINILENV (SEQ ID NO: 93)
>DBYI01000091_51
[Ruminococcus flavefaciens ]
MELSKLELINVYNNCYFI SLWVLNSLTDGEKLDKYKLNNRIANVVNDSQGYFSGKIADEVFDKCSLLFDITPDKTFI SRNKVP IPTCFTVIERIYIKSLINSKYGKLFLSPKEAEEIISCLGDVPDVPINDYLISLPSRTYDYSDKYINNVRFLLMAIKENKEIIYS NKTKEIVHKNKHGYPIRIEYSALYDLFQLSLWSSEGNRPVKINLHSIYGINLTGNVWGEKKSPIEMMETKRCQEPIVIEISNDN NTLEPANILFSMYNTETEKLKNGTYRKKLYYYYFDENEIVNSIFSFGPYVKVISPTVIVDKIKEKIISLSSISNIL (SEQ ID NO: 670)
>ODAI011611274_5
[gut metagenome]
MKLFHKYYSRKLLFAIEVLDALQGAKEQTLNWGELTRLSNRLGMTADLRAEVLNVLTEESRIVRVEDTSNYRLDTSWTTTTPKL PTSKIEEDYLQMILRLPQAEQFLSRELRDRLTDPQASILNTDAIQTIEPNGEQTQLKLSQPEFRMILDAIEMGCAIRYRYISEQ GKAAMEKHAVPWRLQYSAFDNRWWIILYTLKDHRCVKIALGSISDVQLEKHIQVKEADILKAREKDLAAEPAILQVKNTKNALE RCFFVMDRQQFEDSELLEDGSAKLTYRYYHFETSDLLRRLLYLGPAVALIGPPKLRKALLEHVERALNHFRAEA ( SEQ ID NO: 671)
Table 7. Amino acid Sequences of Motifs in Type VI-D CRISPR-Cas Effector Proteins
>MOTIF_l
RXXXXH (SEQ ID NO: 94)
>MOTIF_2
DXXXXQXXXXJLDXXK (SEQ ID NO: 95)
>MOTIF_3
FXXXXXXXXXGXXXXXJR (SEQ ID NO: 96)
>MOTIF_4
KEXNXXXXXXXXXXXNI (SEQ ID NO: 97)
>MOTIF_5
YXXXRXKBLXXXXLF (SEQ ID NO: 98)
>MOTIF_6
DXXXXQXXXXXXDIXK (SEQ ID NO: 672)
>MOTIF_7
KXXKNXGXXXXXLRE (SEQ ID NO: 673)
References
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J.
(1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402.
Bateman, A., Martin, M.J., O'Donovan, C, Magrane, M., Alpi, E., Antunes, R., Bely, B.,
Bingley, M., Bonilla, C, Britto, R., et al. (2017). UniProt: the universal protein knowledgebase. Nucleic Acids Res. 45, D158-D169.
Benson, D.A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, L, Lipman, D.J., Ostell, J., and
Sayers, E.W. (2013). GenBank. Nucleic Acids Res. 41, D36-42.
Eddy, S.R. (2011). Accelerated Profile HMM Searches. PLoS Comput. Biol. 7, el002195.
Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res. 32, 1792-1797.
Edgar, R.C. (2010). Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26, 2460-2461.
Finn, R.D., Bateman, A., Clements, J., Coggill, P., Eberhardt, R.Y., Eddy, S.R., Heger, A.,
Hetherington, K., Holm, L., Mistry, J., et al. (2014). Pfam: the protein families database. Nucleic Acids Res. 42, D222-D230.
Hein, S., Scholz, L, VoB, B., and Hess, W.R. (2013). Adaptation and modification of three
CRISPR loci in two closely related cyanobacteria. RNA Biol. 10, 852-864. Hyatt, D., Chen, G.-L., LoCascio, P.F., Land, M.L., Larimer, F.W., and Hauser, L.J. (2010).
Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119.
Makarova, K.S., Anantharaman, V., Grishin, N.V., Koonin, E.V., and Aravind, L. (2014). CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5.
Peters, J.E., Makarova, K.S., Shmakov, S., and Koonin, E.V. (2017). Recruitment of CRISPR- Cas systems by Tn7-like transposons. Proc. Natl. Acad. Sci. U. S. A. 114, E7358-E7366.
Pruitt, K.D., Tatusova, T., Brown, G.R., and Maglott, D R. (2012). NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy. Nucleic Acids Res. 40, D130-135.
Shmakov, S., Abudayyeh, O.O., Makarova, K.S., Wolf, Y.I., Gootenberg, J.S., Semenova, E., Minakhin, L., Joung, J., Konermann, S., Severinov, K., et al. (2015). Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol. Cell 60, 385— 397.
Shmakov, S., Smargon, A., Scott, D., Cox, D., Pyzocha, N., Yan, W., Abudayyeh, O.O.,
Gootenberg, J.S., Makarova, K.S., Wolf, Y.I., et al. (2017). Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 169-182.
Smargon, A.A., Cox, D.B.T., Pyzocha, N.K., Zheng, K., Slaymaker, I.M., Gootenberg, J.S.,
Abudayyeh, O.A., Essletzbichler, P., Shmakov, S., Makarova, K.S., et al. (2017). Casl3b Is a Type VI-B CRISPR- Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol. Cell 65, 618-630. e7.
Steinegger, M., and Soding, J. (2017). MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets.
Yu, J., Picord, G., Tuffery, P., and Guerois, R. (2015). HHalign-Kbest: exploring sub-optimal alignments for remote homology comparative modeling. Bioinforma. Oxf. Engl. 31, 3850-3852.
Zhu, W., Lomsadze, A., and Borodovsky, M. (2010). Ab initio gene identification in
metagenomic sequences. Nucleic Acids Res. 38, el32-el32.
Example 2. Accelerated in Vivo Functional Screening of Type VI-D CRISPR-Cas Systems
Having identified the minimal suite of Type VI-D CRISPR-Cas system components, we selected two loci for functional validation, those from Eubacterium siraeum DSM 15702
(EsCasl3d) and Ruminococcus sp. N15.MGS-57 (RspCasl3d). RspCasl3d is a member of the largest subgroup of Casl3d proteins which contains 13 of the 31 unique members of the family and shows co-conservation with a putative WYLl accessory protein (FIGs. 1, 6, 7). In contrast, there are no WYL-domain proteins (or other putative accessory proteins) encoded within 3kb of the EsCasl3d effector.
DNA Synthesis and Effector Library Cloning
To test the activity of Type VI-D CRISPR-Cas, we designed and synthesized minimal systems containing RspCasl3d or EsCasl3d into the pET28a(+) vector. The synthesized Ruminococcus sp. RspCasl3d system included RspCasl3d and RspWYLl, codon optimized for E. coli expression under the control of a lac promoter and separated by an E. coli ribosome binding sequence (FIG. 8). Following the open reading frames for RspCasl3d and RspWYLl, we included an acceptor site for a CRISPR array library driven by a J23119 promoter. The Eubacterium siraeum system was prepared similarly but included no gene for a WYL-domain containing protein.
The E. coli codon-optimized genes representing the minimal CRISPR effectors and accessory proteins were synthesized (Genscript) into a custom expression system derived from the pET-28a(+) (EMD-Millipore). Briefly, the Ruminococcus sp. synthesis product included Casl3d and WYLl codon optimized for E. coli expression under the control of a Lac promoter and separated by an E. coli ribosome binding sequence. Following the open reading frames for Casl3d and WYLl, we included an acceptor site for a CRISPR array library driven by a J23119 promoter (Registry of Standard Biological Parts: parts. igem.org/Part:BBa_J23119). Our Eubacterium siraeum system was similarly constructed, but with only the effector protein.
In tandem with the effector gene synthesis, we first computationally designed an oligonucleotide library synthesis (OLS) pool containing "repeat-spacer-repeat" sequences, where "repeat" represents the consensus direct repeat sequence found in the CRISPR array associated with the effector, and "spacer" represents sequences tiling the pACYC184 plasmid. The spacer length was determined by the mode of the spacer lengths found in the endogenous CRISPR array. The repeat-spacer-repeat sequence was appended with restriction sites enabling the bidirectional cloning of the fragment into the aforementioned CRISPR array library acceptor site, as well as unique PCR priming sites to enable specific amplification of a specific repeat-spacer- repeat library from a larger pool. The library synthesis was performed by Agilent Genomics.
We next cloned the repeat-spacer-repeat library into the plasmid containing the minimal engineered locus using the Golden Gate assembly method. In brief, we first amplified each repeat-spacer-repeat from the OLS pool (Agilent Genomics) using unique PCR primers, and pre- linearized the plasmid backbone using Bsal to reduce potential background. Both DNA fragments were purified with Ampure XP (Beckman Coulter) prior to addition to Golden Gate Assembly Master Mix (New England Biolabs) and incubated as per manufacturer's instructions. We further purified and concentrated the Golden Gate reaction to enable maximum
transformation efficiency in the subsequent steps of the bacterial screen.
Accelerated Functional Screening for Casl3d
To accelerate functional screening of Type VI-D systems, we developed a strategy to derive the following functional information in a single screen: 1) crRNA expression direction and processing, 2) nucleic acid substrate type, and 3) targeting requirements such as protospacer adjacent motif (PAM), protospacer flanking sequence (PFS), or target secondary structure. We designed minimal CRISPR array libraries consisting of two consensus direct repeats, each flanking a unique natural -length spacer sequence targeting either the pACYC184 vector or an absent GFP sequence as a negative control. The CRISPR array libraries for EsCasl3d and RspCasl3d systems consisted of 4549 and 3972 pACYC184-targeting spacers respectively, in addition to 452 and 450 spacers targeting the GFP negative control sequence, respectively. We also designed a bidirectional array library cloning strategy to test both possible CRISPR array expression directions in parallel.
The CRISPR array libraries for RspCasl3d and EsCasl3d were cloned into acceptor sites on respective Type VI-D expression plasmids such that each plasmid contained a single library element and orientation (FIG. 8). The resulting plasmid libraries were transformed with pACYC184 into Stbl3 E. coli using electroporation, yielding a maximum of one plasmid library element per cell. Transformed E. coli cells were plated on bioassay plates containing Kanamycin (selecting for the library plasmid), Chloramphenicol (CAM; selecting for intact pACYC184 CAM expression), and Tetracycline (TET; selecting for intact pACYC184 TET expression), such that interruption of pACYC184 plasmid DNA or antibiotic resistance gene expression by the CRISPR-Cas system results in bacterial cell death. Screens were harvested 12h after plating, and plasmid DNA was extracted (FIG. 9). We PCR amplified the CRISPR array region of the input plasmid library prior to transformation and the output plasmid library after bacterial selection on antibiotic plates.
The plasmid library containing the distinct repeat-spacer-repeat elements and Cas proteins was electroporated into Endura electrocompetent E. coli (Lucigen) using a Gene Pulser Xcell® (Bio-rad) following the protocol recommended by Lucigen. The library was either co- transformed with purified pACYC184 plasmid, or directly transformed into pACYC184- containing Endura electrocompetent E. coli (Lucigen), plated onto agar containing
Chloramphenicol® (Fisher), Tetracycline (Alfa Aesar), and Kanamycin (Alfa Aesar) in
BioAssay® dishes (Thermo Fisher), and incubated for 10-12h. After estimation of approximate colony count to ensure sufficient library representation on the bacterial plate, the bacteria were harvested and DNA plasmid extracted using a QIAprep Spin Miniprep® Kit (Qiagen) to create the "output library." By performing a PCR using custom primers containing barcodes and sites compatible with Illumina sequencing chemistry, we generated a barcoded next generation sequencing library from both the pre-transformation "input library" and the post-harvest "output library," which were then pooled and loaded onto a Nextseq 550 (Illumina) to evaluate the effectors. At least two independent biological replicates were performed for each screen to ensure consistency.
Bacterial Screen Sequencing Analysis
Next generation sequencing data for screen input and output libraries were demultiplexed using Illumina bcl2fastq. Reads in resulting fastq files for each sample contained the CRISPR array elements for the screening plasmid library. The direct repeat sequence of the CRISPR array was used to determine the array orientation, and the spacer sequence was mapped to the source plasmid pACYC184 or negative control sequence (GFP) to determine the corresponding target. For each sample, the total number of reads for each unique array element (ra) in a given plasmid library was counted and normalized as follows: (ra+l) / total reads for all library array elements. The depletion score was calculated by dividing normalized output reads for a given array element by normalized input reads.
To identify specific parameters resulting in enzymatic activity and bacterial cell death, we used next generation sequencing (NGS) to quantify and compare the representation of individual CRISPR arrays (i.e., repeat-spacer-repeat) in the PCR of the input and output plasmid libraries. We defined the array depletion ratio as the normalized output read count divided by the normalized input read count. An array was considered to be strongly depleted if the depletion ratio was less than 0.1 (more than 10-fold depletion). When calculating the array depletion ratio across biological replicates, we took the maximum depletion ratio value for a given CRISPR array across all experiments (i.e. a strongly depleted array must be strongly depleted in all biological replicates). We generated a matrix including array depletion ratios and the following features for each spacer target: target strand, transcript targeting, ORI targeting, target sequence motifs, flanking sequence motifs, and target secondary structure. We investigated the degree to which different features in this matrix explained target depletion for RspCasl3d and EsCasl3d systems, thereby yielding a broad survey of functional parameters within a single screen.
Distribution of Bacterial Screening Targets Indicates That Casl3d Targets ssRNA Transcripts
To identify the targeted substrate for Casl3d, we first identified a set of minimal CRISPR arrays that were strongly depleted in 2 screen biological replicates. For both RspCasl3d and EsCasl3d systems, these strongly depleted arrays primarily targeted pACYC184, with minimal depletion of the negative control (FIGs. 10 and 11). We observed 1119 and 806 strongly depleted arrays for the RspCasl3d and EsCasl3d systems, respectively (FIGs. 12A-B). The spatial distribution and strand preference of the strongly depleted target sites along pACYC184 (FIGs. 13A-B) indicate a preference for transcript targeting, suggesting that Casl3d targets single-stranded RNA transcripts. Additionally, the presence of strongly depleted targets within the non-coding region of pACYC184 between the Tet and CAM ORFs corresponds to the extension of RNA transcripts coding for these genes beyond the end of the open reading frame.
These results indicate that targeting of non-essential regions of transcripts might trigger additional catalytic activities of Casl3d enzymes resulting in toxicity and cell death
Lack of PFS for Casl3d and a New Model for Analysis of Sequence Constraints
Previous RNA targeting CRISPR-Cas systems from subtypes VI-A-C have shown varying dependence on a protospacer flanking sequence (PFS) for efficient RNA targeting (Abudayyeh et al., 2016, 2017; Cox et al., 2017; East-Seletsky et al., 2016, 2017; Gootenberg et al., 2017; Smargon et al., 2017). Here we present evidence that RspCasl3d and EsCasl3d have no such flanking sequence requirements. For each enzyme, WebLogos® (Crooks et al., 2004) show that at each of 30 positions before and after the target sequences for strongly depleted arrays the nucleotide frequencies do not appreciably differ from a uniform distribution (FIGs. 14A-B)
To investigate possible flanking sequence requirements further, we developed a combinatorial model to search for up to 3 nucleotide locations distributed across the target or flanking sequences that might explain the observed strongly depleted arrays. We calculated a bit score to measure the degree to which the selected locations correspond to strongly biased outcomes (e.g. all hits or all non-hits). More specifically, we defined a targeting requirement to comprise a set of locations relative to a target sequence and the corresponding nucleotide sequences at those locations. For a given targeting requirement, we define the hit ratio {hr) as the ratio of the number of strongly depleted CRISPR arrays to the total number of library targets satisfying the requirement. When searching for a PAM or PFS of length k, we consider potential targeting requirement locations, where n = spacer length + 2 · flank length. The bit score for a potential targeting requirement is calculated as bitscore =∑— hr log(/ir) over all nucleotide sequences at the specified targeting requirement locations. For CRISPR-Cas systems with known PAM or PFS requirements, such as BzCasl3b, high bit scores for targeting requirements of length 2 or 3 within 15 nt flanks of the target were obtained, and accurately recapitulate the location of the known PFS (FIG. 14C). Conversely, for RspCasl3d and
EsCasl3d, our analysis shows no evidence of flanking or spacer sequences contributing to the targeting efficiency of strongly depleted arrays (FIG. 14C).
Explaining Strongly Depleted Arrays for RspCasl3d and EsCasl3d
Cumulatively, transcript targeting explained 86% and 66% of the strongly depleted arrays for RspCasl3d and EsCasl3d, respectively (FIG. 15). Accordingly, little if any targeting was observed for the ORF template strand. Non-coding and origin of replication (ORI) targeting correspond to actively transcribed regions of the ORI and the extension of coding transcripts into the intergenic region, as corroborated by RNA sequencing of Stbl3 E. coli containing
pACYC184 (FIGs. 14A-B). Secondary structure analysis of the transcripts further enhanced the explanation of targeting for Casl3d. We predicted RNA secondary structure (Lorenz et al., 2011) for all sub-sequences within 30nt of transcript target sites, and found that sequences with no predicted stable secondary structure corresponded to a higher percentage of strongly depleted targets (FIGs. 16A-B). Accordingly, we selected several sub-sequence ranges around the target site (FIGs. 16A-B), and defined a minimal secondary structure targeting requirement to be satisfied if the target site exhibited no predicted stable secondary structure for any of the selected sequence ranges. Among the transcript target sites that satisfy the minimal secondary structure requirement, we can explain 93% and 84% of all strongly depleted arrays for RspCasl3d and EsCasl3d, respectively (FIG. 16C). Together, our results indicate that RspCasl3d and
EsCasl3d are RNA-targeting effectors with no flanking sequence requirements and a preference for minimal secondary structure for RNA targeting in E. coli.
RNA-Sequencing Mature crRNA from In Vivo Bacterial Screen
Sequencing the small RNA from the in vivo bacterial screen began by extracting total RNA from harvested screen bacteria using the Direct-zol RNA MiniPrep® Plus w/ TRI Reagent (Zymo Research). Ribosomal RNA was removed using a Ribo-Zero® rRNA Removal Kit for Bacteria, followed by cleanup using a RNA Clean and Concentrator-5 kit. The resultant ribosomal RNA depleted total RNA was treated with T4 PNK, RNA 5' polyphosphatase, prepared for sequencing using the NEBNext® Small RNA Library Prep Set, and analyzed as described above.
We analyzed the pre-crRNA processing in the screen output samples for the direct repeat orientation that demonstrated successful targeting of pACYC184 and identified a mature 53nt crRNA consisting of a 5' direct repeat truncated by 6nt (FIG. 17). The most common spacer length observed for EsCasl3d was 23nt, with length variation between 20nt and 30nt (length of the native spacer for EsCasl3d).
References
Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D.B.T., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., et al. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573.
Abudayyeh, O.O., Gootenberg, J.S., Essletzbichler, P., Han, S., Joung, J., Belanto, J. J., Verdine, V., Cox, D.B.T., Kellner, M.J., Regev, A., et al. (2017). RNA targeting with CRISPR- Casl3. Nature 550, 280-284.
Cox, D.B.T., Gootenberg, J.S., Abudayyeh, O.O., Franklin, B., Kellner, M.J., Joung, J., and Zhang, F. (2017). RNA editing with CRISPR-Casl3. Science 358, 1019-1027. Crooks, G.E., Hon, G., Chandonia, J.-M., and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome Res. 14, 1188-1190.
East-Seletsky, A., O'Connell, M.R., Knight, S.C., Burstein, D., Cate, J.H.D., Tjian, R., and
Doudna, J.A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270-273.
East-Seletsky, A., O'Connell, M.R., Burstein, D., Knott, G.J., and Doudna, J.A. (2017). RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. Mol. Cell 66, 373-383. e3.
Gootenberg, J.S., Abudayyeh, O.O., Lee, J.W., Essletzbichler, P., Dy, A.J., Joung, J., Verdine, V., Donghia, N., Daringer, N.M., Freije, C.A., et al. (2017). Nucleic acid detection with CRISPR-Casl3a/C2c2. Science 356, 438-442.
Lorenz, R., Bernhart, S.H., Honer zu Siederdissen, C, Tafer, H., Flamm, C, Stadler, P.F., and Hofacker, I.L. (2011). ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26.
Smargon, A.A., Cox, D.B.T., Pyzocha, N.K., Zheng, K., Slaymaker, I.M., Gootenberg, J.S.,
Abudayyeh, O.A., Essletzbichler, P., Shmakov, S., Makarova, K.S., et al. (2017). Casl3b Is a Type VI-B CRISPR- Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Mol. Cell 65, 618-630. e7.
Example 3. Validation of Type VI-D Effector Activity in Vitro (Biochemically)
Effector and Accessory Protein Purification
The effector or accessory protein expression construct was transformed into an E. coli T7 expression strain, NiCo21(DE3)® (New England Biolabs). lmL of overnight culture was inoculated into 1 liter of Luria-Bertani broth growth media (lOg/L tryptone, 5 g/L yeast extract, 5g/L NaCl, Sigma) supplemented with 50 μg/mL Kanamycin. Cells were grown at 37 °C to a cell density of 0.5-0.8 OD6oo. Protein expression was then induced by supplementing with IPTG to a final concentration of 0.2 mM and the culture continued to grow for 14-18 hours at 20°C. The cells were harvested by centrifugation and cell paste was resuspended in 80 ml of freshly prepared Lysis Buffer (50 mM Hepes pH 7.6, 0.5M NaCl, 10 mM imidazole, 14 mM 2- mercaptoethanol and 5% glycerol) supplemented with protease inhibitors (cOmplete, EDTA- free, Roche Diagnostics Corporation). The resuspended cells were broken by passing through a cell disruptor (Constant System Limited). Lysate was cleared by centrifugation twice at 28,000g for 30 min each. The clarified lysate was applied to a 5 ml HisTrap FF chromatography column (GE Life Sciences). Protein purification was performed via FPLC (AKTA Pure, GE Healthcare Life
Sciences). After washing with Lysis Buffer, protein was eluted with a gradient of 10 mM to 250 mM of imidazole. Fractions containing protein of the expected size were pooled, concentrated in Vivaspin 20 ultrafiltration unit (Sartorius) and either used directly for biochemical assays or frozen at -80°C for storage. Protein purity was determined by SDS-PAGE analysis and protein concentration was determined by Qubit® protein assay kit (Thermo Fisher). FIG. 17 shows a Coomassie blue stained polyacrylamide gel of the purified recombinant proteins EsCasl3d, RspCasl3d, and RspWYLl respectively. crRNA and Substrate RNA Preparation
DNA oligo templates for crRNA and substrate RNA in vitro transcription were ordered from IDT (TABLES 8 and 9). Templates for crRNAs were annealed to a short T7 primer (final concentrations 4μΜ) and incubated with T7 RNA polymerase overnight at 37°C using the HiScribe® T7 Quick High Yield RNA Synthesis kit (New England Biolabs). Annealing was performed by incubating T7 primer with templates for 2 minutes at 95°C followed by a -5°C/s ramp down to 23°C. Templates for substrate RNA were PCR amplified to yield dsDNA and then incubated with T7 RNA polymerase at 37°C overnight using the same T7 Quick High Yield RNA Synthesis kit. After in vitro transcription, samples were treated with DNase I (Zymo Research) and then purified using RNA Clean & Concentrator kit (Zymo Research).
5' end labeling was accomplished using the 5' end labeling kit (VectorLabs) and with a IR800® dye-maleimide probe (LI-COR Biosciences). Body labeling of RNA was performed during in vitro transcription using the HiScribe® T7 Quick High Yield RNA Synthesis kit (New England Biolabs). The in vitro transcription reactions contained 2.5 mM Fluorescein- 12-UTP (Sigma Aldrich). Labeled RNA was purified to remove excess dyes using RNA Clean &
Concentrator kit (Zymo Research). The RNA concentration was measured on Nanodrop® 2000 (Thermo Fisher).
The effectors were then incubated with their respective in vitro transcribed pre-crRNAs consisting of a minimal CRISPR array with the repeat-spacer-repeat construction used in the bacterial screening library, but with a single spacer instead of a library. Pre-crRNA cleavage assays were performed at 37°C in processing buffer (20 mM Tris pH8.0, 50 mM KC1, 1 mM EDTA, lOmM MgC12, and 100 ug/ml BSA) unless otherwise indicated, with a final reaction concentration of 200nM of pre-crRNA and varying enzyme concentrations and EDTA as indicated. Reactions were incubated for 30 minutes, and quenched with the addition of lug/uL of proteinase K (Ambion) incubated for 10 minutes at 37°C. Afterwards, 50mM of EDTA was added to the reaction, which was then mixed with equal parts 2x TBE-Urea Sample Buffer (Invitrogen) prior to denaturing at 65C for 3 minutes. Samples were analyzed by denaturing gel electrophoresis on 15% TBE-Urea gels (Invitrogen) and stained using SYBR Gold nucleic acid stain (Invitrogen) for 10-20 minutes prior to imaging on a Gel Doc EZ (Biorad). We found that EsCasl3d and RspCasl3d effectors process pre-crRNAs to form mature crRNAs in the absence of any accessory proteins (FIGs. 20A-D).
RNA-Sequencing of In Vitro Cleaved Pre-crRNA
Sequencing of in vitro cleaved pre-crRNA began with performing and quenching the cleavage assays as described above. The reactions were then column purified using a RNA Clean and Concentrator-5 kit (Zymo Research). The RNA samples were then PNK treated for 3 hours without ATP to enrich for 3'-P ends, after which ATP was added and the reaction incubated for another hour to enrich for 5' -OH ends. The samples were then column purified, incubated with RNA 5' polyphosphatase (Lucigen) and column purified again prior to preparation for next-generation sequencing using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs). The library was paired-end sequenced on a Nextseq 550® (Illumina), and the resulting paired end alignments were analyzed using Geneious 11.0.2 (Biomatters).
Performing next-generation sequencing of the in vitro cleaved RNA fragments enabled the exact identification of the processing intermediates and mature crRNA (FIG. 19) visualized by denaturing gel. For both EsCasl3d and RspCasl3d, sequencing the mature crRNA
corroborated the 6nt truncation from the 5' end of the first direct repeat found in the in vivo small RNA sequencing. For the 3' end, 6 nt of the second direct repeat remained attached to the 3' end of the spacer, yielding a total product of 66nt consistent with the mature crRNA visualized by denaturing gel. The difference between the well-defined 3' end of the mature crRNA forms observed in vitro versus the various lengths identified in vivo may be the result of further truncation in vivo by endogenous RNases following the initial pre-crRNA cleavage. The effector's ability to cleave pre-crRNA at the same location relative to the predicted stem loop structure of either direct repeat (FIG. 19 intermediates 1 and 2) indicates that the Type VI-D CRISPR-Cas effectors are able to process pre-crRNAs containing multiple DRs and spacers.
Effect of EOT A on crRNA Processing
We next examined the dependence of pre-crRNA cleavage on divalent metal ions. We observed that the generation of mature crRNA for both EsCasl3d and RspCasl3d is substantially inhibited by the addition of EDTA (FIGs. 20A-D), while Casl3a from Leptotrichia wadei (LwaCasl3a) is still able to generate mature crRNAs in the presence of EDTA (FIG. 21). This dependence of Casl3d on divalent cations to generate mature crRNA is a notable functional distinction from Casl3a crRNA processing (East-Seletsky et al., 2016; Knott et al., 2017).
Validation of ssRNA Cleavage Activities
We next sought to biochemically validate the RNA-guided ssRNA cleavage activities of the Casl3d enzymes observed in our bacterial screens. Target cleavage assays were performed at 37°C in cleavage buffer (20 mM HEPES pH 7.1, 50 mM KCl, 5 mM MgCk and 5% glycerol). Cas 13 -crRNA complex formation was performed in cleavage buffer by incubating a 2: 1 molar ratio of protein to crRNA at 37°C for 5 minutes, and RspWYLl was added to the Cas 13 -crRNA pre-incubation according to the experimental conditions. For the cleavage reactions at different Cas 13 concentrations, the pre-formed Cas 13 -crRNA complexes were diluted on ice, keeping the Casl3-crRNA ratio constant at 2: 1. The 5' IR800 labeled target ssRNA and/or additional unlabeled and fluorescent body-labeled ssRNAs were then added to the pre-formed complex and incubated at 37°C for 30 minutes. The final concentration of short substrate RNAs was ΙΟΟηΜ and the fluorescent body-labeled ssRNA for collateral effect visualization was 50nM, unless otherwise indicated. Reactions were quenched by adding lug/uL of proteinase K (Ambion) and incubating for 10 minutes at 37°C.
Afterwards, 50mM of EDTA was added to the reaction, which was then mixed with equal parts 2x TBE-Urea Sample Buffer (Invitrogen) prior to denaturing at 65°C for 3 minutes.
Samples were analyzed by denaturing gel electrophoresis on 6% or 15% TBE-Urea gels
(Invitrogen). Fluorescence images were obtained using a Gel Doc EZ® (Biorad), and near- infrared images were obtained using an Odyssey® CLx scanner (LI-COR Biosciences).
Afterwards, the gels were stained for 10-20 minutes using SYBR Gold nucleic acid stain (Invitrogen) and imaged on the Gel Doc EZ® to verify the results from the fluorescence and IR images.
We titrated Apo EsCasl3d and RspCasl3d (100 - 0.4nM) over a non-targeted ssDNA substrate (ΙΟΟηΜ), with the denaturing gel (FIGs. 22A-B) showing minimal cleavage products. We then titrated EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over non- targeted ssDNA substrates (ΙΟΟηΜ), with the resulting denaturing gel (FIGs. 23A-B) showing minimal cleavage products.
We identified spacer sequences for several strongly depleted arrays from bacterial screens for each CRISPR-Cas system and generated pre-crRNAs with the repeat-spacer-repeat arrangement for each effector. We then titrated EsCasl3d and RspCasl3d in complex with crRNA (100 - 0.4nM) over targeted ssDNA substrates (ΙΟΟηΜ), with the resulting denaturing gel (FIGs. 24A-B) showing saturation of target cleavage activity at approx. 50nM RspCasl3d- crRNA complex and ΙΟΟηΜ EsCasl3d-crRNA complex. In an additional experiment, we targeted EsCasl3d and RspCasl3d enzyme-crRNA complexes to 130nt ssRNA substrates containing target sequences complementary to the crRNA spacer and demonstrated targeted RNA cleavage activity for both enzymes (FIGs. 25A-B).
To evaluate the collateral RNA cleavage activity, identical reactions were prepared and supplemented with 800nt fluorescent body -labeled ssRNA fragments that did not contain the target sequence. Both EsCasl3d and RspCasl3d showed substantial collateral activity that occurs with the target cleavage (FIGs. 26A-B). We further demonstrated that both EsCasl3d and RspCasl3d show robust sequence-specific targeted and collateral RNA cleavage activities across multiple crRNAs with and without complementary substrates (FIGs. 26C-D).
Table 8. ssRNA Oligos Used in This Study
Figure imgf000173_0001
Table 9. ssDNA Primers Used to Generate the ssRNA Targets Using in Vitro Transcription
Figure imgf000174_0001
Figure imgf000175_0001
Notes:
IDT IVT: ssDNA primers from IDT were directly annealed with the T7_primer and transcribed
PCR IVT: a PCR using the IDT oligo or GFP as a template was used first to create the dsDNA with the T7 promoter sequence, on which IVT was then performed
IDT: primers ordered from Integrated DNA Technologies
References
East-Seletsky, A., O'Connell, M.R., Knight, S.C., Burstein, D., Cate, J.H.D., Tjian, R., and Doudna, J. A. (2016). Two distinct RNase activities of CRISPR-C2c2 enable guide- RNA processing and RNA detection. Nature 538, 270-273.
Knott, G.J., East-Seletsky, A., Cofsky, J.C., Holton, J.M., Charles, E., O'Connell, M R., and Doudna, J. A. (2017). Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas 13a enzyme. Nat. Struct. Mol. Biol. 24, 825-833.
Example 4. Validation of Type VI-D CRISPR-Cas Systems Comprising Casl3d and WYL1 Activity in Vitro (Biochemically)
Putative accessory proteins containing WYL domains and additional predicted DNA- binding domains are present in the great majority of the Type VI-D loci (FIG. 1). We initially synthesized and screened the predicted minimal CRISPR-Cas system for RspCasl3d including both the RspCasl3d effector and RspWYLl accessory protein. To investigate the modulation of Casl3d by WYL1, we screened both the RspCasl3d effector and RspWYLl accessory protein separately. Comparison of screening results for RspCasl3d effector alone versus the RspCasl3d system, including RspWYLl, shows that RspCasl3d targeted RNA cleavage is increased in the presence of RspWYLl (FIGs. 27A-B). Bacterial screening with RspWYLl alone yielded a minimal number of hits, indicating that RspWYLl has no individual activity (FIG. 28). Cumulatively, these results suggest that RspCasl3d enzymatic activity is modulated either directly or indirectly by WYL1.
We further investigated whether WYL1 could modulate RspCasl3d in vitro by purifying recombinant RspWYLl for use in ssRNA cleavage biochemical assays. To enable high resolution of enhanced or decreased complex activity in the presence of WYL, we selected doses of Casl3d-crRNA complex resulting in approximately 50% cleavage of the target substrates based on a dose titration curve (FIGs. 24A-B). We pre-incubated Casl3d- crRNA with no RspWYLl, an equimolar ratio of RspWYLl to Casl3d, or a molar excess of RspWYLl over Casl3d, and the resulting samples were incubated with target and collateral ssRNA under the same conditions as in the target cleavage assays. We observed that RspWYLl increases both the targeted and collateral ssRNA cleavage activity of RspCasl3d in a dose-dependent manner, with a molar excess of RspWYLl yielding the greatest increase in Casl3d activity (FIGs. 29A-C).
Given that Type VI-D CRISPR-Cas systems appear to have acquired WYL-domain containing accessory proteins on multiple, independent occasions (FIGs. 1, 6, 8, 9), we tested the specificity of RspWYLl in modulating the cleavage activity of orthologous Casl3d effectors. We observed that RspWYLl enhanced the targeted and collateral ssRNA nuclease activities of EsCasl3d to a similar extent as observed for RspCasl3d (FIG. 30A-B). Thus, the effects of WYL1 orthologs appear not to be limited to their native effectors, but instead reflect a modular regulatory mechanism for Casl3d effectors.
To test whether RspWYLl could modulate the activity of a type VI-B Casl3b effector, in vitro ssRNA cleavage biochemical assays were performed using recombinant RspWYLl and Bergeyella zoohelcum Casl3b (BzCasl3b). As shown in FIG. 31, RspWYLl enhanced the activity of BzCasl3b, demonstrating that this accessory protein is also capable of enhancing the activity of Casl3b effectors.
Example 5. Type VI-D CRISPR-Cas Systems can be used with a Fluorescent Reporter for the Specific Detection of Nucleic Acid Species
The dual nuclease activities of Casl3 effectors {i.e., target-specific and non-specific collateral RNase activity) make these effectors promising candidates for use in the detection of nucleic acid species. Some of these methods have been previously described (see, e.g., East-Seletsky et al. (2016), Gootenberg et al. (2017), and Gootenberg et al. (2018)
"Multiplexed and portable nucleic acid detection platform with Casl3, Casl2a, and Csm6" Science 15 Feb 2018: eaaq0179), describing the general principle of RNA detection using Casl3a (East-Seletsky et al. (2016)), supplemented by amplification to increase the detection sensitivity and optimization of additional Casl3a enzymes (Gootenberg et al. (2017)), and most recently, the inclusion of additional RNA targets, orthologous and paralogous enzymes, and Csm6 activator to enable multiplexed detection of nucleic acids along with an increase in detection sensitivity (Gootenberg et al. (2018)). The addition of Casl3d to this toolkit not only provides an additional channel of orthogonal activity for nucleic acid detection, but the nuclease activity-enhancing effect of the WYL1 proteins across orthologous and paralogous effectors suggests that WYL1 proteins can play an activity-enhancing role.
We tested the ability of EsCasl3d or RspCasl3d to cleave RNaseAlert® v2 (Thermo Fisher) substrate under different buffer conditions. Using a buffer of 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg/ml BSA, pH 7.9 provided key improvements from the described cleavage or processing buffers in the following: 1) maximum differentiation of targeting vs. non-targeting, 2) total fluorescence signal intensity, and 3) sufficient stability to support enzyme activity for the duration of the measurement.
We next tested different short fluorescent-quencher RNA substrates for the fluorescent detection of the collateral effect. These included RNase alert v2, a poly-G, and a poly-U substrate. We performed this experiment using a final reaction concentration of 40 nM of the Casl3d effector, 20 nM of crRNA, 5 nM of the target or nontarget RNA, and 160 nM of the fluorescent-quencher substrate along with 0.5 of the murine RNase inhibitor (in 50uL) in the optimized buffer condition as described above. The reaction was incubated for 3 hours at 37°C and the fluorescence read out using a Lightcycler 480 II at one-minute intervals. This demonstrated that both RspCasl3d and EsCasl3d can differentiate between a targeting vs. a non-targeting RNA using a poly-U substrate (FIG. 32). Furthermore, the differences between the activity of the two Casl3d effectors on the different substrate identities suggests the possibility of having multiple channels for the reporter.
The methods described above can include additional improvements to increase detection sensitivity. For example, a pre-amplification step of a nucleic acid in the sample (e.g., a target nucleic acid of interest) may be performed. These pre-amplification step can be performed by any method known in the art including, but not limited to, enzymatic methods such as isothermal amplification and recombinase polymerase amplification (RPA), as well as physical enrichment using methods such as immunoprecipitation. Furthermore, for the detection of DNA species, samples including the DNA species may be transcribed to convert the substrate into a Casl3d compatible substrate (e.g., RNA) while amplifying the target. A number of existing methods for nucleic acid enrichment or background amplification suppression can also be performed to increase the sensitivity and specificity of detection.
Example 6. Type VI-D CRISPR-Cas Systems can be used to Provide Genotype-Gated Control of Cell Death or Dormancy
Hybridization of the Type VI-D CRISPR-Cas effector protein and crRNA with an RNA target complementary to the crRNA spacer forms an active complex that may exhibit nonspecific, "collateral" RNase activity. Such collateral RNAse activity can be used to provide genotype-gated control of cell death or dormancy. The dependence of such activity on the presence of a specific RNA target in a cell is valuable since it enables targeting of specific cell populations based on specific underlying transcriptional states or genotypes. Numerous applications exist in both eukaryotic and prokaryotic settings for such control of cell death or dormancy.
For prokaryotic applications, a Type VI-D CRISPR-Cas system (e.g., including a Type VI-D effector and a crRNA) can be delivered (e.g., in vitro or in vivo) in order to induce cell death or dormancy of specific prokaryote populations (e.g., bacterial populations) in a genotype and transcriptome-specific way. For instance, the Type VI-D CRISPR-Cas system can include one or more crRNAs that specifically target a particular prokaryotic genus, species, or strain. This specific targeting has many therapeutic benefits as it may be used to induce death or dormancy of undesireable bacteria (e.g., pathogenic bacteria such as
Clostridium difficile). In addition, the Type VI-D systems provided herein may be used to target prokaryotic cells having specific genotypes or transcriptional states. Within the microbial diversity that colonizes humans, only a small number of bacterial strains can induce pathogenesis. Further, even within pathogenic strains such as Clostridium difficile, not all members of the bacterial population exist continuously in active, disease-causing states. Thus, using RNA-targeting to control the activity of an Type VI-D effector based on the genotype and transcriptional state of a prokaryotic cell allows for specific control of which cells are targeted without disrupting the entire microbiome.
Additionally, bacterial strains can be readily engineered with genetic circuits or environmentally-controlled expression elements to generate genetic kill switches that limit the growth, colonization, and/or shedding of the engineered bacterial strains. For example, the expression of a TypeVI-D effectors, specific crRNA, or specific target RNA, can be controlled using promoters derived from the regulatory regions of genes encoding proteins expressed in response to external stimuli, such as cold sensitive proteins (PcspA), heat shock proteins (Hsp), chemically inducible systems (Tet, Lac, AraC). The controlled expression of one or more elements of the Type VI-D system allows for the full functional system to be expressed only upon exposure to an environmental stimulus, which in turn activates the nonspecific RNase activity of the system and thereby induces cell death or dormancy. Kill switches including Casl3d effectors as those described herein may be advantageous over traditional kill switch designs such as toxin/antitoxin systems (e.g., CcdB/CcdA Type II toxin/antitoxin systems), since they are not dependent on relative protein expression ratios which may be affected by leaky expression from a promoter (e.g., an environmental-stimulus dependent promoter), and thus allow for more precise control of the kill-switch.
To assess the ability of Casl3d to directly induce the dormancy or death of bacteria cells upon recognition of a target RNA, a variation of the in vivo functional screening described in Example 2 was performed, in which the antibiotic tetracycline was removed from the culture plate. Removing tetracycline selection meant that the survival of the host E. coli was no longer dependent on the successful natural expression of the tetracycline resistance protein by pACYC184. However, the targeting library still contained crRNAs with spacers to the tetracycline resistance gene, TcR. When the dependence of E. coli survival on successful TcR expression is removed, one would expect that there would be no impact on E. coli survival if the Casl3d effector directly cleaved TcR mRNA, and thus no TcR targeting spacers should register as strong depletion event on the in vivo screen. Nevertheless, the screening data without tetracycline selection still showed strongly depleted spacers on the TcR gene (FIGs. 33A-B, 34A-B), suggesting that the effect of Casl3d targeting RNA alone can mediate a growth disadvantage or cell death, even without antibiotic selection.
For eukaryotic applications, many diseases result from specific genotypes or transcriptional states in the diseased cells that distinguish them from healthy cells. Disease related genotypes are often contained in regions of the genome that are expressed, generating transcripts that can be targeted by a Type VI-D effector using a crRNA that specifically targets the genotype. Such targeting can provide cell dormancy or cell death in a population of cells with a specific disease related mutations. An examplary application is the targeted depletion of cancer cells containing specific mutations, such as driver mutations that occur spontaneously in the tumor microenvironment. In addition, the Type VI-D CRISPR-Cas systems described herein can be used as kill-switch mechanisms to induce the death or dormancy of recombinant eukaryotic cells, such as chimeric antigen receptor-expressing T- cells, to limit their activity in inappropriate environments or when no longer desired.
Additionally, in a therapeutic context, numerous disease processes often involve dysregulation of cellular pathways that result in transcriptional states that are different from the normal baseline. A Type VI-D CRISPR-Cas system can be used to specifically induce the death or dormancy of cells that have an altered transcriptome. For example, the system can be used to induce the death or dormancy of cells having a temporally altered
transcriptome, such as cells involved in an anti-inflammatory response during an autoimmune disease flare that are differentiated from normal cells.
The expression of the Type VI-D CRISPR-Cas systems described herein can be controlled and expressed using synthetic biology to induced or trigger cell death or dormancy. For example, the expression of genes encoding each of the components of the Type VI-D CRISPR-Cas systems can be controlled using genetic elements including, but are not limited to, promoters that are regulated by environmental stimuli, such as hypoxia (hif), neuronal activity (fos, arc), heat-shock (HSF-1), or exogenous controls such as light (FixJ), steroids (LexA), alcohol (AlcA), tetracycline (Tet). These promoters can be used to control the expression of components of the Type VI-D CRISPR-Cas system and/or of a specific RNA target to activate the system, thereby inducing the death or dormancy of targeted cells in response to the particular environmental stimuli to which the promoters respond.
Example 7. Adaptation of Type VI-D CRISPR Cas System Effectors for Eukaryotic and Mammalian Activity
Beyond the biochemical and diagnostic applications described herein, programmable RNA-modifying CRISPR-Cas systems such as Type VI-D, e.g., Cas 13d, systems described herein have important applications in eukaryotic cells, ranging from therapeutic uses such as disease transcript correction, to research and development advances, such as for
transcriptome engineering and RNA visualization.
To develop Type VI-D CRISPR Cas systemsfor eukaryotic applications, the constructs encoding the protein effectors are first codon-optimized for expression in mammalian cells, and specific localization tags are optionally appended to either or both the N-terminus or C-terminus of the effector protein. These localization tags can include sequences such as nuclear localization signal (NLS) sequences, which localize the effector to the nucleus for modification of nascent RNAs, as well as nuclear export signal (NES) sequences, which target the effector to the cytoplasm in order to modify mature RNAs.
These sequences are described above in the "Functional Mutations" section. Other accessory proteins, such as fluorescent proteins, may be further appended. It has been demonstrated that the addition of robust, "superfolding" proteins such as superfolding green fluorescent protein (GFP) can increase the activity of Cas 13 enzymes in mammalian cells when appended to the effector (Abudayyeh et al. (2017) Nature 550(7675): 280-4, and Cox et al. (2017) Science 358(6366): 1019-27).
The codon-optimized sequence coding for the Casl3d effector and appended accessory proteins and localization signals is then cloned into a eukaryotic expression vector with the appropriate 5' Kozak eukaryotic translation initiation sequence, eukaryotic promoters, and polyadenylation signals. In mammalian expression vectors, these promoters can include, e.g., general promoters such as CMV, EFla, EFS, CAG, SV40, and cell-type specific RNA polymerase II promoters such as Syn and CamKIIa for neuronal expression, and thyroxine binding globulin (TBG) for hepatocyte expression to name a few. Similarly, useful polyadenylation signals include, but are not limited to, SV40, hGH, and BGH. For expression of the pre-crRNA or mature crRNA, RNA polymerase III promoters such as HI or U6 can be used.
Depending on the application and mode of packaging, the eukaryotic expression vector can be a lentiviral plasmid backbone, adeno-associated viral (AAV) plasmid backbone, or similar plasmid backbone capable of use in recombinant viral vector production. Notably, the small size of Type VI-D CRISPR Cas effector proteins, e.g., Casl3d effector proteins, make them ideally suited for packaging along with its crRNA and appropriate control sequences into a single adeno-associated virus particle; the packaging size limit of 4.7kb for AAV may preclude the use of larger Cas 13 effectors.
After adapting the sequences, delivery vectors, and methods for eukaryotic and mammalian use, different Cas 13d constructs as described herein are characterized for performance. For efficient testing of the mammalian activity levels of various constructs, we use a dual-luciferase reporter expressing both Gaussia luciferase (Glue) and Cypridinia luciferase (Clue) (Abudayyeh et al. (2017) Nature 550(7675): 280-4). Targeting the Glue transcript and comparing the relative activity versus the internal control of the Clue activity enables an estimation of Cas 13d effectiveness in a mammalian context. This activity is corroborated on the reporter through knockdown of endogenous transcripts, such as from the well-characterized KRAS genetic locus. The dual-luciferase reporter construct along with plasmids expressing the type VI-D CRISPR-Cas system and cognate crRNA are delivered using transient transfection (e.g., Lipofectamine® 2000) into model cell lines such as HEK 293T cells.
In addition to testing various construct configurations and accessory sequences on individual targets, pooled library-based approaches are used to determine 1) any targeting dependency of specific Cas 13d effector proteins in mammalian cells as well as 2) the effect of mismatch locations and combinations along the length of the targeting crRNA. Briefly, the pooled library includes a plasmid that expresses a target RNA containing different flanking sequences as well as mismatches to the guide or guides used in the screening experiment, such that the successful target recognition and cleavage results in depletion of the sequence from the library. Furthermore, mRNA sequencing can be used to determine the off- target RNA cleavage effects of the type VI-D CRISPR-Cas system.
Complementary to the possibilities of transcriptome modification using the RNA cleavage activity of Cas 13d, we can also explore the applications of catalytically-inactive Cas 13d effector proteins in which the conserved residues of the two HEPN domains are mutated from the arginine and histidine to alanine. Like other Cas 13 enzymes, catalytically inactive Casl3d (known as dCasl3d) likely will retain its programmable RNA binding activity, though it will no longer be able to cleave target or collateral RNA.
In addition to direct uses of dCasl3d such as in RNA immunoprecipitation, transcript labeling (when dCasl3d effector is fused with fluorescent protein), and translation modification through site-specific targeted disruption of native translational machinery, other domains can be appended onto the dCasl3d protein to provide further functionality.
Activities of these domains include, but are not limited to, RNA base modification (ADAR1, ADAR2, APOBEC), RNA methylation (m6A methyltransferases and demethylases), splicing modifiers (hnRNPAl), localization factors (KDEL retention sequence, mitochondrial targeting signal, peroxisomal targeting signal), translation modification factors (EIF4G translation initiation factor, GLD2 poly(A) polymerase, transcriptional repressors).
Additionally, domains can be appended to provide additional control, such as light-gated control (cryptochromes) and chemically inducible components (FKBP-FRB chemically inducible dimerization).
Optimizing the activity of such fusion proteins requires a systematic way of comparing linkers that connect the dCasl3d with the appended domain. These linkers may include, but are not limited to, flexible glycine-serine (GS) linkers in various combinations and lengths, rigid linkers such as the alpha-helix forming EAAAK (SEQ ID NO: 124) sequence, XTEN linker (Schellenberger V, et al. Nat. Biotechnol. 2009;27: 1186-1190), as well as different combinations thereof (see TABLE 10). The various designs are then assayed in parallel over the same crRNA target complex and functional readout to determine which one yields the desired properties.
For adapting Casl3d for use in targeted RNA base modification (see, e.g., Cox DBT et al, Science 2017 10.1126/science.aaq0180), we begin with the Casl3d ortholog and NES combination that yielded the highest endogenous mammalian RNA knockdown activity and mutate the conserved residues of the two HEPN domains to create a catalytically inactive enzyme. Next, a linker is used to create the fusion protein between Casl3d-NES and the base editing domain. Initially, this domain will consist of the ADAR2DD(E488Q/T375G) mutant engineered previously for hyperactivity and greater specificity when used with Casl3b in REPAIRv2, but alternate deaminases such as ADAR1 and APOBEC 1, among others, can be engineered and assayed in parallel (TABLE 10). Given the likely structural differences between the smaller Casl3d versus the previously characterized Casl3 effectors, alternate linker designs and lengths may yield the optimal design of the base editing fusion protein.
To evaluate the activity of the dCasl3d-derived base editors, the HEK 293T cells are transiently transfected with the dCasl3d-ADAR construct, a plasmid expressing the crRNA, and optionally, a reporter plasmid if targeting the reporter and not an endogenous locus. The cells are harvested 48 hours after transient transfection, the the RNA is extracted and reverse- transcribed to yield a cDNA library that is prepared for next generation sequencing. Analysis of the base composition of loci of samples containing the targeting vs. negative control non- targeting crRNAs provide information about the editing efficiency, and analysis of broader changes to the transcriptome will yield information about the off-target activity.
One particular advantage of developing an RNA base editing system using Casl3d is that the small size, on average 20% smaller than the existing Casl3 effectors, enables more ready packaging in AAV of dCasl3d-ADAR along with its crRNA and control elements without the need for protein truncations. This all-in-one AAV vector enables greater efficacy of in vivo base editing in tissues, which is particularly relevant as a path towards therapeutic applications of Casl3d. In base editing and other applications, the small size, the lack of a biochemical PFS, and robust activity of Casl3d effectors make it a valuable addition to the toolbox of programmable RNA modifying enzymes.
Multiplexing of Casl3d with multiple crRNAs targeting different sequences enables the manipulation of multiple RNA species for therapeutic applications requiring manipulation of multiple transcripts simultaneously.
Table 10. Amino Acid Sequences of Motifs and Functional Domains in Engineered Variants ofType VI-D CRISPR-Cas Effector Proteins
>LINKER_1
GS
>LINKER_2
GSGGGGS ( SEQ ID NO: 125)
>LINKER_3
GGGGSGGGGSGGGGS (SEQ ID NO: 126)
>LINKER_4
GGSGGSGGSGGSGGSGGS (SEQ ID NO: 127)
[ADAR1, ADAR2 : C-term fusion (or optionally N-term) ]
>ADAR1DD-WT
SLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPC GDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGEGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLG LQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNW CLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYG NWISKPQEEKNF (SEQ ID NO: 128)
>ADAR1DD-E1008Q (Cox et al . , 2017)
SLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPC GDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLG LQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNW CLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYG NWISKPQEEKNF (SEQ ID NO: 129)
>ADAR2 DD-WT
QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGWMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAE IISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKAR GQLRTKIESGEGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNWGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSR AMYQRI SNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRV HGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT (SEQ ID NO: 130)
>ADAR2 DD-E488Q (Cox et al., 2017) QLHLPQVLADAVSRLVLGKFGDLTDNFSSPHARRKVLAGWMTTGTDVKDAKVISVSTGTKCINGEYMSDRGLALNDCHAE IISRRSLLRFLYTQLELYLNNKDDQKRSIFQKSERGGFRLKENVQFHLYISTSPCGDARIFSPHEPILEEPADRHPNRKAR GQLRTKIESGQGTIPVRSNASIQTWDGVLQGERLLTMSCSDKIARWNWGIQGSLLSIFVEPIYFSSIILGSLYHGDHLSR AMYQRI SNIEDLPPLYTLNKPLLSGISNAEARQPGKAPNFSVNWTVGDSAIEVINATTGKDELGRASRLCKHALYCRWMRV HGKVPSHLLRSKITKPNVYHESKLAAKEYQAAKARLFTAFIKAGLGAWVEKPTEQDQFSLT ( SEQ ID NO: 131)
[Cytidine deaminase, AID, APOBEC1: N-term fusion (or optionally C-term) ]
>AID-AP0BEC1 (Dickerson et al . , 2003, Komor et al . , 2017)
MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYWKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWF TSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAW EGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL ( SEQ ID NO: 132)
>Lamprey_AID-APOBECl (Rogozin et al . , 2007, Komor et al . , 2017)
MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEI FSIRKVEEYLRD NPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQI GLWNLRDNGVGLNVMVSEHYQCCRKI FIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 133)
>APOBECl_BEl (Komor et al., 2016)
MSSETGPVAVDPTLRRRI EPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTR CSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPS NEAHWPRYPHLWVRLYVLELYCII LGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 134)
References
Abudayyeh, O.O., Gootenberg, J.S., Essletzbichler, P., Han, S., Joung, J., Belanto, J.J.,
Verdine, V., Cox, D.B.T., Kellner, M.J., Regev, A., et al. (2017). RNA targeting with CRISPR-Casl3. Nature 550, 280-284.
Cox, D.B.T., Gootenberg, J.S., Abudayyeh, O.O., Franklin, B., Kellner, M.J., Joung, J., and Zhang, F. (2017). RNA editing with CRISPR-Casl3. Science 358, 1019-1027.
Schellenberger V., Wang C.W., Geething N.C, Spink, B.J., Campbell, A., To, W., Scholle, M.D., Yin, Y., Yao, Y., Bogin, O., et al. (2009). A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol 2009; 27: 1186-1190.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:
1. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) system comprising:
an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and
a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2, wherein the effector protein is capable of binding to the RNA guide and of targeting the target nucleic acid sequence complementary to the RNA guide spacer sequence.
2. The system of claim 1, wherein the effector protein comprises an amino acid sequence provided in Table 2.
3. The system of claim 1, wherein the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
4. The system of any one of claims 1-3, wherein the effector protein comprises at least two HEPN domains, wherein none, one, or two of the HEPN domains are catalytically deactivated.
5. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) system comprising:
an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid;
a CRISPR-associated protein or a nucleic acid encoding the CRISPR-associated protein; and
an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein comprises:
i) at least one WYL domain, wherein the WYL domain comprises an amino acid sequence PXXXiXXXXXXXXXYL (SEQ ID NO: 198), wherein Xi is C, V, I, L, P, F, Y, M, or W, and wherein X is any amino acid; and ii) at least one ribbon-ribbon-helix (RHH) fold or at least one helix-turn-helix (HTH) domain;
wherein the CRISPR-associated protein is capable of binding to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
6. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) - associated (Cas) system comprising:
an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid;
a CRISPR-associated protein or a nucleic acid encoding the CRISPR-associated protein; and
an accessory protein or a nucleic acid encoding the accessory protein, wherein the accessory protein comprises at least one WYL domain, and wherein the accessory protein comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6;
wherein the CRISPR-associated protein is capable of binding to the RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, and wherein the accessory protein modulates an activity of the CRISPR-associated protein.
7. The system of claim 5 or claim 6, wherein the activity is a nuclease activity.
8. The system of claim 7, wherein the nuclease activity is a DNAse activity.
9. The system of claim 7, wherein the nuclease activity is a targeted RNAse activity or a collateral RNAse activity.
10. The system of any one of claims 5-9, wherein the accessory protein increases the activity of the CRISPR-associated protein.
11. The system of any one of claims 5-9, wherein the accessory protein decreases the activity of the CRISPR-associated protein.
12. The system of any one of claims 6-11, wherein the accessory protein comprises an amino acid sequence provided in any one of Tables 4, 5, and 6.
13. The system of claim 5 or claim 6, wherein the accessory protein is RspWYLl (SEQ ID NO: 81).
14. The system of any one of claims 5-13, wherein the targeting of the target nucleic acid results in a modification of the target nucleic acid.
15. The system of any one of claims 5-14, wherein the CRISPR-associated protein is a Class 2 CRISPR-Cas system protein.
16. The system of any one of claims 5-15, wherein the CRISPR-associated protein comprises a RuvC domain.
17. The system of any one of claims 5-15, wherein the CRISPR-associated protein is selected from the group consisting of a Type VI Cas protein, a Type V Cas protein, and a Type II Cas protein.
18. The system of any one of claims 5-15, wherein the CRISPR-associated protein is a Casl3a protein, a Casl3b protein, a Casl3c protein, a Casl2a protein, or a Cas9 protein.
19. The system of any one of claims 5-15, wherein the CRISPR-associated protein is a Type VI-D CRISPR-Cas effector protein comprising at least two HEPN domains, wherein none, one, or two of the HEPN domains are catalytically deactivated.
20. The system of claim 19, wherein the effector protein comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2.
21. The system of claim 19 or claim 20, wherein the effector protein comprises an amino acid sequence provided in Table 2.
22. The system of any one of claims 19-21, wherein the effector protein is RspCasl3d (SEQ ID NO: 2) or EsCasl3d (SEQ ID NO: 1).
23. The system of any one of claims 1-22, wherein the target nucleic acid is an RNA.
24. The system of any one of claims 1-22, wherein the target nucleic acid is a DNA.
25. The system of any one of claims 1-4 and 14, wherein the modification of the target nucleic acid is a cleavage event.
26. The system of any one of claims 1-4, 14, and 25, wherein the modification results in: (a) decreased transcription; (b) decreased translation; or (c) both (a) and (b), of the target nucleic acid.
27. The system of any one of claims 1-4, 14, and 25, wherein the modification results in (a) increased transcription; (b) increased translation; or (c) both (a) and (b), of the target nucleic acid.
28. The system of any one of claims 4 and 19-22, wherein the effector protein comprises one or more amino acid substitutions within at least one of the HEPN domains.
29. The system of claim 28, wherein the one or more one amino acid substitutions comprise an alanine substitution at an amino acid residue corresponding to R295, H300, R849, or H854 of SEQ ID NO: 1, or R288, H293, R820, or H825 of SEQ ID NO: 2.
30. The system of claim 28 or claim 29, wherein the one or more amino acid substitutions result in a reduction of a nuclease activity of the Type VI-D CRISPR-Cas effector protein, as compared to the nuclease activity of the Type VI-D CRISPR-Cas effector protein without the one or more acid substitutions.
31. The system of any one of claims 1-30, wherein the direct repeat sequence comprises a nucleotide sequence provided in Table 3.
32. The system of any one of claims 1-30, wherein the direct repeat sequence comprises 5' - X1X2X3X4TX5TX6AAAC-3' (SEQ ID NO: 199) at the 3' terminal end of the RNA guide, and wherein Xi is A or C or G, X2 is A or G or T, X3 is A or G or T, X4 is C or G or T, X5 is
Figure imgf000190_0001
33. The system of any one of claims 1-30, wherein the direct repeat sequence comprises either 5'-CACCCGTGCAAAATTGCAGGGGTCTAAAAC-3' (SEQ ID NO: 152) or 5'- CACTGGTGCAAATTTGCACTAGTCTAAAAC-3' (SEQ ID NO: 153).
34. The system of any one of claims 1-33, wherein the spacer comprises from about 15 to about 42 nucleotides.
35. The system of any one of claims 1-34, wherein the RNA guide further comprises a trans-activating CRISPR RNA (tracrRNA).
36. The system of any one of claims 1-35, further comprising a single-stranded donor template or a double-stranded donor template.
37. The system of claim 36, wherein the donor template is a DNA or an RNA.
38. The system of any one of claims 1-37, further comprising a target RNA or a nucleic acid encoding the target RNA, wherein the target RNA comprises a sequence that is capable of hybridizing to the spacer sequence of the RNA guide.
39. The system of any one of claims 1-38, wherein the system is present in a delivery system.
40. The system of claim 39, wherein the delivery system comprises a delivery vehicle selected from the group consisting of a nanoparticle, a liposome, an adeno-associated virus, an exosome, a microvesicle, and a gene-gun.
41. A cell comprising the system of any one of claims 1-40.
42. The cell of claim 41, wherein the cell is a eukaryotic cell.
43. The cell of claim 42, wherein the eukaryotic cell is a mammalian cell or a plant cell.
44. The cell of claim 41, wherein the cell is a prokaryotic cell.
45. The cell of claim 44, wherein the prokaryotic cell is a bacterial cell.
46. An animal model or a plant model comprising the cell of any one of claims 41-45.
47. A method of cleaving a target nucleic acid, the method comprising contacting the target nucleic acid with a system of any one of claims 1-40;
wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid;
wherein the CRISPR-associated protein or the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex;
wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and
wherein upon binding of the complex to the target nucleic acid sequence the CRISPR- associated protein or the Type VI-D CRISPR effector protein cleaves the target nucleic acid.
48. The method of claim 47, wherein the target nucleic acid is within a cell.
49. A method of inducing dormancy or death of a cell, the method comprising contacting the cell with a system of any one of claims 1-40;
wherein the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid;
wherein the CRISPR-associated protein or the Type VI-D CRISPR effector protein associates with the RNA guide to form a complex;
wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and
wherein upon binding of the complex to the target nucleic acid sequence the CRISPR- associated protein or the Type VI-D CRISPR-Cas effector protein cleaves a non-target nucleic acid within the cell, thereby inducing dormancy or death of the cell.
50. The method of any one of claims 47-49, wherein the target nucleic acid is an RNA selected from the group consisting of an mRNA, a tRNA, a ribosomal RNA, a non-coding RNA, a lncRNA, or a nuclear RNA.
51. The method of claim 49, wherein the target nucleic acid is a DNA selected from the group consisting of chromosomal DNA, mitochondrial DNA, single-stranded DNA, or plasmid DNA.
52. The method of any one of claims 47-51 , wherein upon binding of the complex to the target nucleic acid, the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein exhibits collateral RNAse activity.
53. The method of any one of claims 49-52, wherein the death is via apoptosis, necrosis, necroptosis, or a combination thereof.
54. The method of any one of claims 48-53, wherein the cell is a cancer cell.
55. The method of claim 54, wherein the cancer cell is a tumor cell.
56. The method of any one of claims 48-53, wherein the cell is an infectious agent cell or a cell infected with an infectious agent.
57. The method of claim 48-53, wherein the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.
58. A method of treating a condition or disease in a subject in need thereof, the method comprising administering to the subject a system of any one of claims 1-40,
wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease;
wherein the CRISPR-associated protein or the Type VI-D CRISPR-Cas effector protein associates with the RNA guide to form a complex;
wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and
wherein upon binding of the complex to the target nucleic acid sequence the CRISPR- associated protein or the Type VI-D CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject.
59. The method of claim 58, wherein the condition or disease is a cancer or an infectious disease.
60. The method of claim 59, wherein the condition or disease is cancer, and wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a
neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non- Hodgkin's lymphoma, and urinary bladder cancer.
61. A system according to any one of claims 1-40, for use in a method selected from the group consisting of RNA sequence specific interference; RNA sequence-specific gene regulation; screening of RNA, RNA products, IncRNA, non-coding RNA, nuclear RNA, or mRNA; mutagenesis; inhibition of RNA splicing; fluorescence in situ hybridization;
breeding; induction of cell dormancy; induction of cell cycle arrest; reduction of cell growth and/or cell proliferation; induction of cell anergy; induction of cell apoptosis; induction of cell necrosis; induction of cell death; or induction of programmed cell death.
62. The system of claim 1, wherein the effector protein is fused to a base-editing domain, a RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
63. The system of claim 5 or claim 6, wherein the CRISPR-associated protein is fused to a base-editing domain, a RNA methyltransferase, a RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
64. The system of claim 62 or claim 63, wherein the base editing domain is selected from the group consisting of Adenosine Deaminase Acting on RNA (ADAR) 1 (ADAR1), ADAR2, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC), and activation-induced cytidine deaminase (AID).
65. The system of any one of claims 1-40, further comprising an RNA-binding fusion polypeptide that comprises an RNA-binding domain and a base-editing domain.
66. The system of claim 65, wherein the base-editing domain is selected from the group consisting of ADAR1, ADAR2, APOBEC, and AID.
67. The system of claim 65 or claim 66, wherein the RNA-binding domain is MS2.
68. A method of modifying an RNA molecule, comprising contacting the RNA molecule with a system according to any one of claims 62-67.
69. A method of detecting a target RNA in a sample, the method comprising:
a) contacting the sample with:
(i) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to the target RNA;
(ii) a Type VI-D CRISPR-Cas effector protein or a nucleic acid encoding the effector protein; and
(iii) a labeled detector RNA;
wherein the effector protein associates with the RNA guide to form a complex; wherein the RNA guide hybridizes to the target RNA; and wherein upon binding of the complex to the target RNA, the effector protein exhibits collateral RNAse activity and cleaves the labeled detector RNA; and
b) measuring a detectable signal produced by cleavage of the labeled detector RNA, wherein said measuring provides for detection of the target RNA in the sample.
70. The method of claim 69, wherein the effector protein comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in Table 2.
71. The method of claim 69 or claim 70, wherein the target RNA is single-stranded.
72. The method of any one of claims 69-71 , wherein the target RNA was transcribed from a DNA molecule.
73. The method of any one of claims 69-72, further comprising contacting the sample with an accessory protein comprising at least one WYL domain.
74. The method of claim 73, wherein the accessory protein comprises an amino acid sequence having at least 85% sequence identity to an amino acid sequence provided in any one of Tables 4, 5, and 6.
75. The method of any one of claims 69-74, further comprising comparing the detectable signal with a reference signal and determining the amount of target RNA in the sample.
76. The method of any one of claims 69-75, wherein the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing.
77. The method of any one of claims 69-76, wherein the labeled detector RNA comprises a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluor pair.
78. The method of any one of claims 69-77, wherein upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is decreased.
79. The method of any one of claims 69-78, wherein upon cleavage of the labeled detector RNA by the effector protein, an amount of detectable signal produced by the labeled detector RNA is increased.
80. The method of any one of claims 69-79, wherein the labeled detector RNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.
81. The method of any one of claims 69-80, wherein a detectable signal is produced when the labeled detector RNA is cleaved by the effector protein.
82. The method of any one of claims 69-81, further comprising pre-amplify a nucleic acid in the sample prior to the contacting step.
PCT/US2018/040649 2017-06-30 2018-07-02 Novel crispr rna targeting enzymes and systems and uses thereof Ceased WO2019006471A2 (en)

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110066865A (en) * 2019-04-30 2019-07-30 常州桐树生物科技有限公司 A kind of detection method and probe of the nucleic acid specific fragment based on CRISPR-Cas13a
US10822617B2 (en) 2018-06-08 2020-11-03 Locana, Inc. RNA-targeting fusion protein compositions and methods for use
WO2021007529A1 (en) 2019-07-10 2021-01-14 Locanabio, Inc. Rna-targeting knockdown and replacement compositions and methods for use
WO2021076060A1 (en) * 2019-10-18 2021-04-22 Nanyang Technological University Programmable rna editing platform
US11098297B2 (en) 2017-06-09 2021-08-24 Editas Medicine, Inc. Engineered Cas9 nucleases
US11168322B2 (en) 2017-06-30 2021-11-09 Arbor Biotechnologies, Inc. CRISPR RNA targeting enzymes and systems and uses thereof
WO2022119974A1 (en) 2020-12-01 2022-06-09 Locanabio, Inc. Rna-targeting compositions and methods for treating cag repeat diseases
WO2022136370A1 (en) * 2020-12-22 2022-06-30 Helmholtz Zentrum Muenchen - Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh) Application of crispr/cas13 for therapy of rna virus and/or bacterium induced diseases
US11453891B2 (en) 2017-05-10 2022-09-27 The Regents Of The University Of California Directed editing of cellular RNA via nuclear delivery of CRISPR/CAS9
WO2022221278A1 (en) 2021-04-12 2022-10-20 Locanabio, Inc. Compositions and methods comprising hybrid promoters
US11499151B2 (en) 2017-04-28 2022-11-15 Editas Medicine, Inc. Methods and systems for analyzing guide RNA molecules
WO2022256440A2 (en) 2021-06-01 2022-12-08 Arbor Biotechnologies, Inc. Gene editing systems comprising a crispr nuclease and uses thereof
US11667903B2 (en) 2015-11-23 2023-06-06 The Regents Of The University Of California Tracking and manipulating cellular RNA via nuclear delivery of CRISPR/CAS9
WO2023096584A3 (en) * 2021-11-25 2023-06-22 Casbio (S) Pte Ltd Novel crispr/cas13 systems and uses thereof
WO2023154807A2 (en) 2022-02-09 2023-08-17 Locanabio, Inc. Compositions and methods for modulating pre-mrna splicing
EP4025588A4 (en) * 2019-09-05 2023-09-06 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
WO2023205637A1 (en) 2022-04-18 2023-10-26 Locanabio, Inc. Rna-targeting compositions and methods for treating c9/orf72 diseases
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
WO2024173645A1 (en) 2023-02-15 2024-08-22 Arbor Biotechnologies, Inc. Gene editing method for inhibiting aberrant splicing in stathmin 2 (stmn2) transcript
US12110545B2 (en) 2017-01-06 2024-10-08 Editas Medicine, Inc. Methods of assessing nuclease cleavage
US12201699B2 (en) 2014-10-10 2025-01-21 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
WO2025072446A1 (en) * 2023-09-27 2025-04-03 Keith Brown Cell specific activation
US12286727B2 (en) 2016-12-19 2025-04-29 Editas Medicine, Inc. Assessing nuclease cleavage
US12338436B2 (en) 2018-06-29 2025-06-24 Editas Medicine, Inc. Synthetic guide molecules, compositions and methods relating thereto
US12351818B2 (en) 2022-11-23 2025-07-08 Amber Bio Inc. Gene-modifying endonucleases
WO2025171210A1 (en) 2024-02-09 2025-08-14 Arbor Biotechnologies, Inc. Compositions and methods for gene editing via homology-mediated end joining
WO2026015832A2 (en) 2024-07-12 2026-01-15 Arbor Biotechnologies, Inc. Reverse transcriptases and gene editing systems comprising such
WO2026015829A2 (en) 2024-07-12 2026-01-15 Arbor Biotechnologies, Inc. Small reverse transcriptases and gene editing systems comprising such

Families Citing this family (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10876101B2 (en) 2017-03-28 2020-12-29 Locanabio, Inc. CRISPR-associated (Cas) protein
US20210139890A1 (en) * 2017-06-30 2021-05-13 Arbor Biotechnologies, Inc. Novel crispr rna targeting enzymes and systems and uses thereof
US10476825B2 (en) 2017-08-22 2019-11-12 Salk Institue for Biological Studies RNA targeting methods and compositions
US11970720B2 (en) 2017-08-22 2024-04-30 Salk Institute For Biological Studies RNA targeting methods and compositions
WO2019204369A1 (en) * 2018-04-17 2019-10-24 Applied Stemcell, Inc. Compositions and methods for treating spinal muscular atrophy
AU2019265019B2 (en) 2018-05-11 2025-11-06 Beam Therapeutics Inc. Methods of substituting pathogenic amino acids using programmable base editor systems
SG11202012015YA (en) * 2018-06-08 2021-01-28 Locanabio Inc Compositions and methods for the modulation of adaptive immunity
EP3847254A4 (en) 2018-09-07 2022-08-10 Beam Therapeutics Inc. Compositions and methods for delivering a nucleobase editing system
WO2020051562A2 (en) 2018-09-07 2020-03-12 Beam Therapeutics Inc. Compositions and methods for improving base editing
AU2020208346B2 (en) * 2019-01-14 2026-01-29 University Of Rochester Targeted nuclear RNA cleavage and polyadenylation with CRISPR-cas
US20220145297A1 (en) * 2019-01-29 2022-05-12 The Regents Of The University Of California Rna-targeting cas enzymes
AU2020215730A1 (en) 2019-01-31 2021-07-29 Beam Therapeutics Inc. Nucleobase editors having reduced non-target deamination and assays for characterizing nucleobase editors
WO2020168088A1 (en) * 2019-02-13 2020-08-20 Beam Therapeutics Inc. Compositions and methods for treating glycogen storage disease type 1a
JP7693552B2 (en) * 2019-02-13 2025-06-17 ビーム セラピューティクス インク． Adenosine deaminase base editors and methods for using same to modify nucleobases in target sequences
CN120174005A (en) 2019-02-13 2025-06-20 比姆医疗股份有限公司 Modified immune cells with adenosine deaminase base editors for modifying nucleobases in target sequences
EP3994691B1 (en) 2019-07-03 2024-05-29 Hewlett-Packard Development Company, L.P. Audio signal dereverberation
EP3996738A4 (en) * 2019-07-11 2023-07-26 Arbor Biotechnologies, Inc. Novel crispr dna targeting enzymes and systems
WO2021011504A1 (en) * 2019-07-12 2021-01-21 Duke University Nanoparticle systems for targeted delivery of crispr/cas13 and methods of using same
WO2021011493A1 (en) * 2019-07-12 2021-01-21 Duke University 3' utr crispr-dcas 13 engineering system and methods of using same
JP7759311B2 (en) 2019-07-19 2025-10-23 フラッグシップ パイオニアリング イノベーションズ シックス，エルエルシー Recombinase compositions and methods of use
US20220267773A1 (en) * 2019-07-23 2022-08-25 University Of Rochester Targeted RNA cleavage with CRISPR-Cas
JP2022546701A (en) * 2019-08-27 2022-11-07 アーバー バイオテクノロジーズ， インコーポレイテッド Novel CRISPR DNA targeting enzymes and systems
EP4028047A4 (en) 2019-09-09 2023-07-26 Arbor Biotechnologies, Inc. NOVEL CRISPR-DNA TARGETED ENZYMES AND SYSTEMS
MX2022002919A (en) * 2019-09-10 2022-09-09 Science Solutions Llc Novel class 2 type ii and type v crispr-cas rna-guided endonucleases.
CN115175996A (en) * 2019-09-20 2022-10-11 博德研究所 Novel type VI CRISPR enzymes and systems
CN110551812B (en) * 2019-09-26 2020-08-18 暨南大学 CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof
EP4034138A4 (en) 2019-09-27 2024-07-31 Beam Therapeutics, Inc. COMPOSITIONS AND METHODS FOR TREATING BLOOD CANCER
JP7680440B2 (en) * 2019-10-15 2025-05-20 エージェンシー フォー サイエンス， テクノロジー アンド リサーチ Assays for measuring nucleic acid modifying enzyme activity
US11443235B2 (en) 2019-11-14 2022-09-13 International Business Machines Corporation Identifying optimal weights to improve prediction accuracy in machine learning techniques
US20230022311A1 (en) * 2019-11-26 2023-01-26 New York Genome Center, Inc Methods and compositions involving crispr class 2, type vi guides
CN115315519A (en) 2020-02-28 2022-11-08 辉大(上海)生物科技有限公司 VI-E and VI-F CRISPR-Cas systems and their uses
CN111440826A (en) * 2020-03-16 2020-07-24 中山大学附属第一医院 6-methyladenine-modified CasRx carrier system targeting RNA and preparation method and application
CN111257297B (en) * 2020-03-31 2023-06-16 海南大学 RNA detection method based on Cas14a enzyme
EP4150095A4 (en) * 2020-05-11 2024-06-19 The Regents of the University of Colorado, a body corporate COMPOSITIONS, METHODS AND USES FOR QUANTIFYING TRANSCRIPTION AND BIODETECTION OF SMALL MOLECULES USING A CRISPR-CAS TYPE VI ASSAY
CN111549054A (en) * 2020-05-22 2020-08-18 中国农业科学院植物保护研究所 CRISPR/RfxCas13d anti-plant RNA virus vector and its construction method and application
CA3196831A1 (en) 2020-09-25 2022-03-31 Beam Therapeutics Inc. Fratricide resistant modified immune cells and methods of using the same
CN118109438A (en) * 2020-09-30 2024-05-31 辉大(上海)生物科技有限公司 Engineered CRISPR/Cas13 systems and their uses
EP4240853A2 (en) * 2020-11-03 2023-09-13 Science Solutions LLC Novel class 2 crispr-cas rna-guided endonucleases
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CA3200929A1 (en) * 2020-12-01 2022-06-09 Alexandra HOWELL Compositions and methods for cleaving viral genomes
US20240043840A1 (en) * 2020-12-14 2024-02-08 New York Genome Center, Inc. Chemically modified crispr-cas13 guide rnas
TW202302844A (en) * 2021-03-09 2023-01-16 美商阿伯生物技術公司 Compositions comprising a variant polypeptide and uses thereof
US20230058054A1 (en) * 2021-06-29 2023-02-23 Huigene Therapeutics Co., Ltd. Crispr/cas system and uses thereof
WO2023025103A1 (en) * 2021-08-22 2023-03-02 Huigene Therapeutics Co., Ltd. Crispr-cas13 system for treating sod1-associated diseases
CN114438055B (en) * 2021-10-26 2022-08-26 山东舜丰生物科技有限公司 Novel CRISPR enzymes and systems and uses
CN116949011A (en) * 2022-04-26 2023-10-27 中国科学院动物研究所 Isolated Cas13 protein, gene editing system based on same and use thereof
WO2024196814A1 (en) 2023-03-17 2024-09-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods for treatment of age-related macular degeneration
WO2025042559A1 (en) * 2023-08-21 2025-02-27 The Regents Of The University Of California Methods and compositions for modulating bacteriophage nucleic acids
US20250163395A1 (en) * 2023-11-21 2025-05-22 Roche Molecular Systems, Inc. Systems and methods of novel crispr-cas proteins for in-vitro applications
CN118581277A (en) * 2024-06-18 2024-09-03 四川农业大学 A method for detecting porcine circovirus type 2 based on CRISPR-Cas13d technology

Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993024641A2 (en) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Adeno-associated virus with inverted terminal repeat sequences as promoter
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5593972A (en) 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
WO2014093622A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
WO2015054653A2 (en) 2013-10-11 2015-04-16 Massachusetts Eye & Ear Infirmary Methods of predicting ancestral virus sequences and uses thereof
WO2015070083A1 (en) 2013-11-07 2015-05-14 Editas Medicine,Inc. CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS
EP3009511A2 (en) 2015-06-18 2016-04-20 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2016094872A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Dead guides for crispr transcription factors
WO2016094874A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Escorted and functionalized guides for crispr-cas systems
WO2016205764A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017070605A1 (en) 2015-10-22 2017-04-27 The Broad Institute Inc. Type vi-b crispr enzymes and systems
US20170362644A1 (en) 2016-06-16 2017-12-21 The Regents Of The University Of California Methods and compositions for detecting a target rna
WO2017219027A1 (en) 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013266968B2 (en) 2012-05-25 2017-06-29 Emmanuelle CHARPENTIER Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
JP2018513681A (en) 2015-03-31 2018-05-31 エクセリゲン サイエンティフィック, インコーポレイテッドExeligen Scientific, Inc. Cas9 retroviral integrase and Cas9 recombinase system for targeted integration of DNA sequences into the genome of a cell or organism
US20180305689A1 (en) * 2015-04-22 2018-10-25 Mina Therapeutics Limited Sarna compositions and methods of use
GB201602473D0 (en) 2016-02-11 2016-03-30 Horizon Discovery Ltd Replicative transposon system
EP3500967A1 (en) 2016-08-17 2019-06-26 The Broad Institute, Inc. Methods for identifying class 2 crispr-cas systems
US11104937B2 (en) * 2017-03-15 2021-08-31 The Broad Institute, Inc. CRISPR effector system based diagnostics
US11174515B2 (en) * 2017-03-15 2021-11-16 The Broad Institute, Inc. CRISPR effector system based diagnostics
US10876101B2 (en) 2017-03-28 2020-12-29 Locanabio, Inc. CRISPR-associated (Cas) protein
EP3638789A4 (en) 2017-06-12 2021-03-10 California Institute of Technology RNA CONDITIONAL GUIDES
US10392616B2 (en) 2017-06-30 2019-08-27 Arbor Biotechnologies, Inc. CRISPR RNA targeting enzymes and systems and uses thereof
US20210139890A1 (en) 2017-06-30 2021-05-13 Arbor Biotechnologies, Inc. Novel crispr rna targeting enzymes and systems and uses thereof
US10476825B2 (en) * 2017-08-22 2019-11-12 Salk Institue for Biological Studies RNA targeting methods and compositions

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993024641A2 (en) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Adeno-associated virus with inverted terminal repeat sequences as promoter
US5593972A (en) 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
WO2014093622A2 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications
US8795965B2 (en) 2012-12-12 2014-08-05 The Broad Institute, Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
US8871445B2 (en) 2012-12-12 2014-10-28 The Broad Institute Inc. CRISPR-Cas component systems, methods and compositions for sequence manipulation
WO2015054653A2 (en) 2013-10-11 2015-04-16 Massachusetts Eye & Ear Infirmary Methods of predicting ancestral virus sequences and uses thereof
WO2015070083A1 (en) 2013-11-07 2015-05-14 Editas Medicine,Inc. CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS
WO2016094872A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Dead guides for crispr transcription factors
WO2016094874A1 (en) 2014-12-12 2016-06-16 The Broad Institute Inc. Escorted and functionalized guides for crispr-cas systems
EP3009511A2 (en) 2015-06-18 2016-04-20 The Broad Institute, Inc. Novel crispr enzymes and systems
US20160208243A1 (en) 2015-06-18 2016-07-21 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2016205764A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Novel crispr enzymes and systems
EP3009511B1 (en) 2015-06-18 2017-05-31 The Broad Institute, Inc. Novel crispr enzymes and systems
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
WO2017070605A1 (en) 2015-10-22 2017-04-27 The Broad Institute Inc. Type vi-b crispr enzymes and systems
US20170362644A1 (en) 2016-06-16 2017-12-21 The Regents Of The University Of California Methods and compositions for detecting a target rna
WO2017219027A1 (en) 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems

Non-Patent Citations (69)

* Cited by examiner, † Cited by third party
Title
ABUDAYYEH ET AL., NATURE, vol. 550, no. 7675, 2017, pages 280 - 4
ABUDAYYEH, O.O.; GOOTENBERG, J.S.; ESSLETZBICHLER, P.; HAN, S.; JOUNG, J.; BELANTO, J.J.; VERDINE, V.; COX, D.B.T.; KELLNER, M.J.;: "RNA targeting with CRISPR-Casl3", NATURE, vol. 550, 2017, pages 280 - 284
ABUDAYYEH, O.O.; GOOTENBERG, J.S.; KONERMANN, S.; JOUNG, J.; SLAYMAKER, I.M.; COX, D.B.T.; SHMAKOV, S.; MAKAROVA, K.S.; SEMENOVA,: "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector", SCIENCE, vol. 353, 2016, pages aaf5573, XP055407082, DOI: doi:10.1126/science.aaf5573
ALLERSON ET AL.: "Fully 2 '-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA", J. MED. CHEM., vol. 48.4, 2005, pages 901 - 904, XP002404763, DOI: doi:10.1021/jm049167j
ALTSCHUL, S.F.; MADDEN, T.L.; SCHAFFER, A.A.; ZHANG, J.; ZHANG, Z.; MILLER, W.; LIPMAN, D.J.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
APONTE-UBILLUS ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 102, no. 3, 2018, pages 1045 - 54
BATEMAN, A.; MARTIN, M.J.; O'DONOVAN, C.; MAGRANE, M.; ALPI, E.; ANTUNES, R.; BELY, B.; BINGLEY, M.; BONILLA, C.; BRITTO, R. ET AL: "UniProt: the universal protein knowledgebase", NUCLEIC ACIDS RES., vol. 45, 2017, pages D158 - D169
BELL ET AL.: "A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing", BMC GENOMICS, vol. 15.1, 2014, pages 1002
BENSON, D.A.; CAVANAUGH, M.; CLARK, K.; KARSCH-MIZRACHI, I.; LIPMAN, D.J.; OSTELL, J.; SAYERS, E.W., GENBANK. NUCLEIC ACIDS RES., vol. 41, 2013, pages D36 - 42
BRAMSEN ET AL.: "Development of therapeutic-grade small interfering RNAs by chemical engineering", FRONT. GENET., vol. 3, 20 August 2012 (2012-08-20), pages 154, XP055134928, DOI: doi:10.3389/fgene.2012.00154
CANVER ET AL.: "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis", NATURE, vol. 527, no. 7577, 12 November 2015 (2015-11-12), pages 192 - 7, XP055274680, DOI: doi:10.1038/nature15521
CHEN ET AL.: "Spatially resolved, highly multiplexed RNA profiling in single cells", SCIENCE, vol. 348, no. 6233, 24 April 2015 (2015-04-24), pages aaa6090, XP055391215, DOI: doi:10.1126/science.aaa6090
COOPER ET AL.: "RNA and disease", CELL, vol. 136.4, 2009, pages 777 - 793
COX DBT ET AL., SCIENCE, 2017
COX ET AL., SCIENCE, vol. 358, no. 6366, 2017, pages 1019 - 27
COX, D.B.T.; GOOTENBERG, J.S.; ABUDAYYEH, O.O.; FRANKLIN, B.; KELLNER, M.J.; JOUNG, J.; ZHANG, F.: "RNA editing with CRISPR-Casl", SCIENCE, vol. 358, 2017, pages 1019 - 1027, XP055491658, DOI: doi:10.1126/science.aaq0180
COX, D.B.T.; GOOTENBERG, J.S.; ABUDAYYEH, O.O.; FRANKLIN, B.; KELLNER, M.J.; JOUNG, J.; ZHANG, F.: "RNA editing with CRISPR-Casl3", SCIENCE, vol. 358, 2017, pages 1019 - 1027, XP055491658, DOI: doi:10.1126/science.aaq0180
CROOKS, G.E.; HON, G.; CHANDONIA, J.-M.; BRENNER, S.E.: "WebLogo: a sequence logo generator", GENOME RES, vol. 14, 2004, pages 1188 - 1190, XP002408756, DOI: doi:10.1101/gr.849004
DATLINGER ET AL.: "Pooled CRISPR screening with single-cell transcriptome read-out", NAT. METHODS., vol. 14, no. 3, March 2017 (2017-03-01), pages 297 - 301
EAST-SELETSKY, A.; O'CONNELL, M.R.; BURSTEIN, D.; KNOTT, G.J.; DOUDNA, J.A.: "RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes", MOL. CELL, vol. 66, 2017, pages 373 - 383
EAST-SELETSKY, A.; O'CONNELL, M.R.; KNIGHT, S.C.; BURSTEIN, D.; CATE, J.H.D.; TJIAN, R.; DOUDNA, J.A.: "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection", NATURE, vol. 538, 2016, pages 270 - 273, XP055407060, DOI: doi:10.1038/nature19802
ECKSTEIN: "Phosphorothioates, essential components of therapeutic oligonucleotides", NUCL. ACID THER., vol. 24, 2014, pages 374 - 387
EDDY, S.R.: "Accelerated Profile HMM Searches", PLOS COMPUT. BIOL., vol. 7, 2011, pages e1002195
EDGAR, R.C.: "MUSCLE: multiple sequence alignment with high accuracy and high throughput", NUCLEIC ACIDS RES., vol. 32, 2004, pages 1792 - 1797, XP008137003, DOI: doi:10.1093/nar/gkh340
EDGAR, R.C.: "Search and clustering orders of magnitude faster than BLAST", BIOINFORMATICS, vol. 26, 2010, pages 2460 - 2461
EPSTEIN; SCHAFFER, MOL. THER., vol. 24, 2016, pages S50
FINN, R.D.; BATEMAN, A.; CLEMENTS, J.; COGGILL, P.; EBERHARDT, R.Y.; EDDY, S.R.; HEGER, A.; HETHERINGTON, K.; HOLM, L.; MISTRY, J.: "Pfam: the protein families database", NUCLEIC ACIDS RES., vol. 42, 2014, pages D222 - D230
GOOTENBERG ET AL.: "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6", SCIENCE, 15 February 2018 (2018-02-15), pages eaaq0179
GOOTENBERG ET AL.: "Nucleic acid detection with CRISPR-Casl3a/C2c2", SCIENCE, vol. 356, no. 6336, 28 April 2017 (2017-04-28), pages 438 - 442, XP055481345, DOI: doi:10.1126/science.aam9321
GOOTENBERG, J.S.; ABUDAYYEH, O.O.; LEE, J.W.; ESSLETZBICHLER, P.; DY, A.J.; JOUNG, J.; VERDINE, V.; DONGHIA, N.; DARINGER, N.M.; F: "Nucleic acid detection with CRISPR-Casl3a/C2c2", SCIENCE, vol. 356, 2017, pages 438 - 442, XP055481345, DOI: doi:10.1126/science.aam9321
GRISSA ET AL., BMC BIOINFORMATICS, vol. 8, 2007, pages 172
GRISSA ET AL., NUCLEIC ACIDS RES, vol. 36, 2008, pages W145 - 8
GRISSA ET AL., NUCLEIC ACIDS RES., vol. 35, 2007, pages W52 - 7
HALLBRINK ET AL.: "Prediction of cell-penetrating peptides", METHODS MOL. BIOL., vol. 1324, 2015, pages 39 - 58
HAMMOND ET AL.: "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae", NAT. BIOTECHNOL., vol. 34, no. 1, January 2016 (2016-01-01), pages 78 - 83, XP055389211, DOI: doi:10.1038/nbt.3439
HEIN ET AL., RNA BIOL., vol. 10, 2013, pages 852 - 64
HEIN, S.; SCHOLZ, I.; VOB, B.; HESS, W.R.: "Adaptation and modification of three CRISPR loci in two closely related cyanobacteria", RNA BIOL, vol. 10, 2013, pages 852 - 864
HIROSAWA ET AL., NUCL. ACIDS RES., vol. 45, no. 13, 2017, pages e1 18
HLAVOVA ET AL.: "Improving microalgae for biotechnology—from genetics to synthetic biology", BIOTECHNOL. ADV., vol. 33, 1 November 2015 (2015-11-01), pages 1194 - 203
HYATT, D.; CHEN, G.-L.; LOCASCIO, P.F.; LAND, M.L.; LARIMER, F.W.; HAUSER, L.J.: "Prodigal: prokaryotic gene recognition and translation initiation site identification", BMC BIOINFORMATICS, vol. 11, 2010, pages 119, XP021071474, DOI: doi:10.1186/1471-2105-11-119
KELLEY ET AL.: "Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing", J. BIOTECHNOL., vol. 233, 10 September 2016 (2016-09-10), pages 74 - 83, XP029674969, DOI: doi:10.1016/j.jbiotec.2016.06.011
KNOTT, G.J.; EAST-SELETSKY, A.; COFSKY, J.C.; HOLTON, J.M.; CHARLES, E.; O'CONNELL, M.R.; DOUDNA, J.A.: "Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme", NAT. STRUCT. MOL. BIOL., vol. 24, 2017, pages 825 - 833
KONERMANN ET AL.: "Optical control of mammalian endogenous transcription and epigenetic states", NATURE, vol. 500.7463, 2013, pages 472
LORENZ, R.; BERNHART, S.H.; HONER ZU SIEDERDISSEN, C.; TAFER, H.; FLAMM, C.; STADLER, P.F.; HOFACKER, I.L.: "ViennaRNA Package 2.0.", ALGORITHMS MOL. BIOL., vol. 6, 2011, pages 26
MAKAROVA, K.S.; ANANTHARAMAN, V.; GRISHIN, N.V.; KOONIN, E.V.; ARAVIND, L.: "CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems", FRONT. GENET., 2014, pages 5
MOLLER; LIANG, PEERJ, vol. 5, 2017, pages e3788
NAKAMURA ET AL., NUCL. ACIDS RES., vol. 28, 2000, pages 292
NICOLAOU ET AL.: "Molecular diagnosis of peanut and legume allergy", CURR. OPIN. ALLERGY CLIN. IMMUNOL., vol. 11, no. 3, June 2011 (2011-06-01), pages 222 - 8
NOWAK ET AL.: "Guide RNA engineering for versatile Cas9 functionality", NUCL. ACID. RES., vol. 44, no. 20, 16 November 2016 (2016-11-16), pages 9555 - 9564
OSBOME ET AL.: "RNA-dominant diseases", HUM. MOL. GENET., vol. 18, no. 8, 15 April 2009 (2009-04-15), pages 1471 - 81
PETERS, J.E.; MAKAROVA, K.S.; SHMAKOV, S.; KOONIN, E.V.: "Recruitment of CRISPR-Cas systems by Tn7-like transposons", PROC. NATL. ACAD. SCI. U. S. A., vol. 114, 2017, pages E7358 - E7366
PRUITT, K.D.; TATUSOVA, T.; BROWN, G.R.; MAGLOTT, D.R.: "NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy", NUCLEIC ACIDS RES., vol. 40, 2012, pages D130 - 135
RAMAKRISHNA ET AL.: "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA", GENOME RES., vol. 24, no. 6, June 2014 (2014-06-01), pages 1020 - 7, XP055128944, DOI: doi:10.1101/gr.171264.113
SCHELLENBERGER V ET AL., NAT. BIOTECHNOL., vol. 27, 2009, pages 1186 - 1190
SCHELLENBERGER V.; WANG C.W.; GEETHING N.C.; SPINK, B.J.; CAMPBELL, A.; TO, W.; SCHOLLE, M.D.; YIN, Y.; YAO, Y.; BOGIN, O. ET AL.: "A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner", NAT BIOTECHNOL, vol. 27, 2009, pages 1186 - 1190
See also references of EP3645722A2
SHMAKOV, S.; ABUDAYYEH, O.O.; MAKAROVA, K.S.; WOLF, Y.I.; GOOTENBERG, J.S.; SEMENOVA, E.; MINAKHIN, L.; JOUNG, J.; KONERMANN, S.;: "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems", MOL. CELL, vol. 60, 2015, pages 385 - 397, XP055481389, DOI: doi:10.1016/j.molcel.2015.10.008
SHMAKOV, S.; SMARGON, A.; SCOTT, D.; COX, D.; PYZOCHA, N.; YAN, W.; ABUDAYYEH, O.O.; GOOTENBERG, J.S.; MAKAROVA, K.S.; WOLF, Y.I.: "Diversity and evolution of class 2 CRISPR-Cas systems", NAT. REV. MICROBIOL., vol. 15, 2017, pages 169 - 182, XP002767857, DOI: doi:10.1038/nrmicro.2016.184
SMARGON, A.A.; COX, D.B.T.; PYZOCHA, N.K.; ZHENG, K.; SLAYMAKER, I.M.; GOOTENBERG, J.S.; ABUDAYYEH, O.A.; ESSLETZBICHLER, P.; SHMA: "Casl3b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28", MOL. CELL, vol. 65, 2017, pages 618 - 630
STEINEGGER, M.; SODING, J., MMSEQS2 ENABLES SENSITIVE PROTEIN SEQUENCE SEARCHING FOR THE ANALYSIS OF MASSIVE DATA SETS, 2017
STEPHEN J. ET AL.: "Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction", NUCL. ACIDS RES., vol. 40.9, 2012, pages e64 - e64, XP055433199, DOI: doi:10.1093/nar/gks028
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 3251 - 60
VERWAAL ET AL.: "CRISPR/Cpfl enables fast and simple genome editing of Saccharomyces cerevisiae", YEAST, 8 September 2017 (2017-09-08)
WEST ET AL., VIROLOGY, vol. 160, 1987, pages 38 - 47
WRIGHT, ADDISON V. ET AL.: "Rational design of a split-Cas9 enzyme complex", PROC. NAT'L. ACAD. SCI., vol. 112.10, 2015, pages 2984 - 2989, XP055283739, DOI: doi:10.1073/pnas.1501698112
YU, J.; PICORD, G.; TUFFERY, P.; GUEROIS, R.: "HHalign-Kbest: exploring sub-optimal alignments for remote homology comparative modeling", BIOINFORMA. OXF. ENGL., vol. 31, 2015, pages 3850 - 3852
ZETSCHE; VOLZ; ZHANG: "A split-Cas9 architecture for inducible genome editing and transcription modulation", NATURE BIOTECH., vol. 33.2, 2015, pages 139 - 142, XP055227889, DOI: doi:10.1038/nbt.3149
ZHONG ET AL., J. GENET. SYNDR. GENE THER., vol. SI, 2012, pages 008
ZHU, W.; LOMSADZE, A.; BORODOVSKY, M.: "Ab initio gene identification in metagenomic sequences", NUCLEIC ACIDS RES., vol. 38, 2010, pages e132 - e132

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