WO2024251229A1 - Cas酶及其系统和应用 - Google Patents

Cas酶及其系统和应用 Download PDF

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Publication number
WO2024251229A1
WO2024251229A1 PCT/CN2024/097935 CN2024097935W WO2024251229A1 WO 2024251229 A1 WO2024251229 A1 WO 2024251229A1 CN 2024097935 W CN2024097935 W CN 2024097935W WO 2024251229 A1 WO2024251229 A1 WO 2024251229A1
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Prior art keywords
sequence
cas enzyme
nucleic acid
cas
cell
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French (fr)
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WO2024251229A9 (zh
Inventor
冯争艳
吴垒磊
毛少帅
杨长青
朱琦
管婧雯
臧赢
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Epigenic Therapeutics Inc
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Epigenic Therapeutics Inc
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Priority to EP24818760.1A priority Critical patent/EP4726035A1/en
Priority to CN202480038064.2A priority patent/CN121358850A/zh
Publication of WO2024251229A1 publication Critical patent/WO2024251229A1/zh
Publication of WO2024251229A9 publication Critical patent/WO2024251229A9/zh
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    • 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]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • 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
    • 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 application relates to the field of biomedicine, and specifically to a Cas enzyme and its system and application.
  • CRISPR-Cas Clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated genes
  • Components of the system involved in host defense include one or more effector proteins capable of modifying DNA or RNA and RNA guide elements responsible for targeting the activity of these proteins to specific sequences on phage DNA or RNA, which can be reprogrammed to target alternative DNA or RNA targets.
  • CRISPR-Cas systems can be roughly divided into two categories: Class 1 systems are composed of multiple effector proteins, and Class 2 systems are composed of a single effector protein, which is complexed with an RNA guide to target a DNA or RNA substrate.
  • Class 1 systems are composed of multiple effector proteins
  • Class 2 systems are composed of a single effector protein, which is complexed with an RNA guide to target a DNA or RNA substrate.
  • the single-subunit effector composition of Class 2 systems provides a simpler set of components for engineering and application conversion, and has been an important source of programmable effectors to date.
  • the characterization and engineering of Class 2 CRISPR-Cas systems, such as CRISPR-Cas9 have paved the way for diverse and extensive biotechnological applications in genome editing and other aspects.
  • nucleic acids and polynucleotides i.e., DNA, RNA, or any hybrid, derivative, or modification thereof.
  • the present application provides an isolated Cas enzyme, the Cas enzyme comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-58 or a sequence having at least about 80% identity with the amino acid sequence as shown in any one of the SEQ ID NOs: 1-58.
  • the Cas enzyme comprises an amino acid sequence as shown in any one of the SEQ ID NOs: 1-58 having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or a sequence having about 100% identity.
  • the present application provides a Cas enzyme comprising an amino acid sequence as shown in the following table:
  • the Cas enzyme has a catalytically active domain capable of binding to a target DNA chain and/or a catalytically active domain capable of cleaving the target DNA chain.
  • the catalytically active domain comprises one or more amino acid changes, so that the Cas enzyme has only the activity of binding to the target DNA chain, or has the activity of binding to the target DNA chain and the activity of cutting the target DNA single strand.
  • the present application provides a fusion molecule, which comprises the Cas enzyme described in the present application and one or more heterologous functional domains.
  • the one or more heterologous functional domains are capable of regulating the expression of one or more gene products.
  • the one or more heterologous functional domains are fused directly or indirectly to the Cas enzyme.
  • the one or more heterologous functional domains are selected from the group consisting of an autohelicase, a nuclease, a helicase-nuclease, a DNA methyltransferase, a DNA hydroxymethylase, a histone methylase, a histone demethylase, a histone acetyltransferase, a histone deacetylase, a phosphatase, a kinase, a transcription (co)activator, a transcription repressor, a DNA binding protein, a DNA structural protein, a marker protein, a reporter protein, a fluorescent protein, a ligand binding protein, a signal peptide, a subcellular localization sequence, an antibody epitope, and an affinity purification tag.
  • an autohelicase a nuclease, a helicase-nuclease, a DNA methyltransferase, a DNA hydroxymethylase,
  • the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, deaminase activity, transcription activation activity, transcription repression activity, transcription release factor activity, reverse transcriptase activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • the present application provides an engineered, programmable, non-naturally occurring CRISPR-Cas system, the system comprising the Cas enzyme described in the present application or the fusion molecule described in the present application, and one or more guide RNAs, wherein the one or more guide RNAs target the loci of nucleic acid molecules encoding one or more gene products in a cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and/or cut the loci of the nucleic acid molecules encoding one or more gene products; and the Cas enzyme or the fusion molecule and the guide RNA do not exist naturally together.
  • the present application provides an engineered, non-naturally occurring vector system, the vector system comprising one or more vectors, the one or more vectors comprising: a) a first regulatory element, the first regulatory element being operably linked to one or more guide RNAs, the one or more guide RNAs being capable of hybridizing with a target sequence in a locus of a nucleic acid molecule encoding one or more gene products, and b) a second regulatory element, the second regulatory element being operably linked to the Cas enzyme described in the present application or the fusion molecule described in the present application, wherein the components a) and b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and/or cut the locus of the nucleic acid molecule encoding one or more gene products; and the Cas enzyme or the fusion molecule
  • expression of the one or more gene products is altered.
  • expression of the gene product is decreased or increased.
  • the gene product is a protein.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell includes, but is not limited to, cells of mice, monkeys, humans, farm animals, sports animals, and pets.
  • the mammalian cell is a human cell.
  • the Cas enzyme is codon-optimized for expression in eukaryotic cells.
  • the guide RNA comprises a guide sequence fused to a tracr sequence.
  • the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to the nucleic acid molecule targeted by the guide RNA.
  • the direct repeat sequence is 10 to 70 nucleotides in length.
  • the direct repeat sequence is 31 to 36 nucleotides in length.
  • the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99, or comprises a nucleotide sequence having at least 95% sequence identity with a nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99.
  • the spacer sequence is 16 to 24 nucleotides in length.
  • the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence that is capable of complementary pairing with the spacer sequence.
  • the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).
  • NLS nuclear localization sequences
  • the system is introduced into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection, and conjugation.
  • the present application provides a method for changing the expression of one or more gene products, the method comprising introducing an engineered, non-naturally occurring CRISPR-Cas system into a cell containing and expressing a nucleic acid molecule encoding the one or more gene products, the system comprising the Cas enzyme described in the present application or the fusion molecule described in the present application, and one or more guide RNAs, the one or more guide RNAs targeting the locus of the nucleic acid molecule encoding the one or more gene products, thereby guiding the Cas enzyme or the fusion molecule to bind to and/or cut the locus, thereby changing the expression of the one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.
  • the present application provides a method for changing the expression of one or more gene products, the method comprising introducing an engineered, non-naturally occurring vector system into a cell containing and expressing a nucleic acid molecule encoding the one or more gene products, the vector system comprising one or more vectors, the one or more vectors comprising: a) a first regulatory element, the first regulatory element being operably linked to one or more guide RNAs, the one or more guide RNAs being capable of hybridizing with a target sequence in the locus of the nucleic acid molecule encoding the one or more gene products, and b) a second regulatory element, the second regulatory element being operably linked to the Cas enzyme described in the present application or the fusion molecule described in the present application, wherein the components a) and b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding the one or more gene products in the cell, thereby guiding the Cas
  • expression of the gene product is decreased or increased.
  • the gene product is a protein.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell includes but is not limited to Limited to cells from mice, monkeys, humans, farm animals, sports animals, and pets.
  • the mammalian cell is a human cell.
  • the Cas enzyme is codon-optimized for expression in eukaryotic cells.
  • the guide RNA comprises a guide sequence fused to a tracr sequence.
  • the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to the nucleic acid molecule targeted by the guide RNA.
  • the direct repeat sequence is 10 to 70 nucleotides in length.
  • the direct repeat sequence is 31 to 36 nucleotides in length.
  • the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99, or comprises a nucleotide sequence having at least 95% sequence identity with a nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99.
  • the spacer sequence is 16 to 24 nucleotides in length.
  • the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence that is capable of complementary pairing with the spacer sequence.
  • the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).
  • NLS nuclear localization sequences
  • the method comprises introducing the CRISPR-Cas system or the vector system into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection, and conjugation.
  • the present application provides a nucleic acid encoding the Cas enzyme described in the present application, the fusion molecule described in the present application, or the CRISPR-Cas system described in the present application.
  • the present application provides a cell, comprising the Cas enzyme described in the present application, the fusion molecule described in the present application, the CRISPR-Cas system described in the present application, the vector system described in the present application and/or the nucleic acid described in the present application.
  • the present application provides a kit comprising the Cas enzyme described in the present application, the fusion molecule described in the present application, the CRISPR-Cas system described in the present application, the vector system described in the present application, the nucleic acid described in the present application and/or the cell described in the present application.
  • the kit further comprises a container for placing the Cas enzyme, the fusion molecule, the CRISPR-Cas system, the vector system, the nucleic acid and/or the cell, and instructions for use.
  • Figure 1A-1B shows the schematic diagram of the assay process and fluorescence results of the cleavage activity of the Cas enzyme described in the present application in eukaryotic cells, as well as the PAM motif required for the gRNA binding of the Cas enzyme.
  • Figures 2A-2B show a schematic diagram of the determination process of the cleavage activity of the Cas enzyme described in the present application in eukaryotic cells and the comparison results of green fluorescent cells of different Cas enzymes.
  • Figures 3A-3B show the schematic diagram of the determination process of the in vitro cleavage activity of the Cas enzyme described in the present application and its PAM sequence and the sequencing results.
  • Figure 4 shows the site information and amplification detection results of detecting the Cas enzyme activity described in the present application at the endogenous site.
  • Figures 5A-5C show the GFP fluorescence results of different variants of the Cas enzyme described in the present application after cleavage activity assay in eukaryotic cells.
  • Figures 6A-6B show the results of the proportion of green fluorescent cells after the cutting activity of sgRNA treated with the Cas enzyme described in this application combined with different DR (Direct repeat) region optimization schemes in eukaryotic cells was measured.
  • FIG. 7 shows the results of the green fluorescent cell ratio after the cleavage activity of the Cas enzyme described in the present application combined with sgRNA of different Spacer lengths in eukaryotic cells was measured.
  • Figures 8A-8B show the results of green fluorescent cell ratios after different variants of the Cas enzyme described in the present application were tested for cleavage activity in eukaryotic cells.
  • Figure 8C shows the results of cleavage activity tests (green fluorescent cell ratios) of different variants of the Cas enzyme described in the present application in different PAM reporter systems.
  • Figures 9A-9C show a schematic diagram of the structure of a cytosine base editor comprising the Cas enzyme described in the present application, as well as the results of detecting the base editing efficiency at two endogenous sites, EMX1 and VEGFA.
  • FIG. 10 shows the activation effect of the gene activation epigenetic tool comprising the Cas enzyme described in the present application on detecting the target site gene expression at the CXCR4 endogenous site.
  • identity can be used interchangeably with “homology”, and it generally refers to the relationship between two or more polypeptide molecules or two or more nucleic acid molecule sequences, and the relationship is determined by comparing their sequences.
  • identity also refers to the degree of nucleic acid molecules or polypeptide sequence correlation, which can be determined by the matching between two or more nucleotides or two or more amino acid sequences.
  • the identity percentage (%) of an amino acid sequence is defined as the percentage of the total number of residues in the candidate sequence that the amino acid residues identical with the amino acid residues in the reference polypeptide sequence are accounted for after the alignment sequence and, if necessary, introducing a gap to reach the maximum percentage sequence identity, and any conservative substitution is not considered as a part of the sequence identity.
  • the comparison for the purpose of measuring the percentage amino acid sequence identity can be achieved in a variety of ways within the art technology, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment sequence, including any algorithm required for reaching the maximum alignment in the full length of the sequence compared.
  • the calculation of the percent identity (%) of a polypeptide molecule or nucleic acid molecule sequence can also determine the total number of residues based on the sequence mutation type.
  • the mutation type includes insertion (extension) at either or both ends of the sequence, deletion (truncation) at either or both ends of the sequence, substitution/replacement of one or more amino acids/nucleotides, insertion within the sequence, and deletion within the sequence.
  • the mutation type is one or more of the following: substitution/replacement of one or more amino acids/nucleotides, insertion within the sequence, and deletion within the sequence
  • the total number of residues is calculated as the larger of the molecules being compared.
  • the mutation type also includes insertion (extension) at either or both ends of the sequence or deletion (truncation) at either or both ends of the sequence or insertion within the sequence and deletion within the sequence
  • the number of amino acids inserted or deleted at either or both ends or within is less than 20
  • the sequences being compared can be aligned in a manner that produces the maximum match between the sequences, and the gaps (if any) in the alignment are resolved by a specific algorithm.
  • catalytically active domain refers to an identifiable or determinable conserved structural entity in a Cas protein (enzyme) that exhibits significant secondary structure content, and the conserved structure is a region in which the Cas protein (enzyme) implements functions such as binding and/or cutting polynucleotides.
  • An exemplary catalytically active domain can be a protease of the Cas9 family, which has two catalytically active domains, one of which is HNH-like, and its function is to cut off a single-stranded polynucleotide (target chain) paired with a guide RNA, and the other domain is RuvC-like, and its function is to cut off the complementary chain of the target chain.
  • binding e.g., with respect to a target DNA binding (catalytically active) domain of a polypeptide or protease
  • macromolecules e.g., between a protein and a nucleic acid
  • association e.g., when molecule X is referred to as interacting with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner.
  • binding interaction components need to be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of the binding interaction may be sequence-specific.
  • fusion molecule generally refers to a molecule consisting of at least two parts (bipartite molecule), which contains the enzyme (protein or peptide) of the present application, which is coupled to at least one other part to form a single entity.
  • the enzyme and the at least one other part can be separated by a linker, or can be directly coupled.
  • the at least one other part can be fused to the enzyme of the present application at any amino acid other than the N-terminus, C-terminus or terminal amino acid.
  • the other part can be fused to the part already contained in the fusion molecule.
  • Those skilled in the art are fully aware of the optimal order and/or combination of determinations for determining the parts in the fusion molecule of the present application.
  • the term does not include such a fusion molecule in which the fusion produces a naturally occurring peptide.
  • heterologous generally refers to a nucleotide or polypeptide sequence that is not present in a natural nucleic acid or protein, respectively.
  • heterologous functional domain may refer to about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains or a part of a fusion molecule described in the present application in addition to the Cas enzyme described in the present application.
  • heterologous functional domains that may be included in the fusion molecule described in the present application or that may be fused to the Cas enzyme described in the present application include, but are not limited to, epitope tags, reporter gene sequences, and one or more protein domains having the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, ⁇ -glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), to include the autofluorescent protein of blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • Cas enzymes can be fused to a gene sequence encoding a protein or protein fragment, which binds to a DNA molecule or binds to other cell molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • GAL4 DNA binding domain fusions GAL4 DNA binding domain fusions
  • HSV herpes simplex virus
  • expression generally refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein.
  • Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
  • polynucleotide generally refer to a polymeric form of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides can have any three-dimensional structure and can perform any function known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined according to connection analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA short hairpin RNA
  • miRNA micro-RNA
  • ribozymes ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleotides
  • plasmids vectors, isolated DNA of any sequence, isolated RNA
  • Polynucleotides can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • nucleic acid molecules, polypeptides, or combinations and systems thereof are used interchangeably.
  • nucleic acid molecules, polypeptides, or combinations and systems thereof they generally mean that the nucleic acid molecules or polypeptides are at least substantially free from at least one other component with which they are associated in nature or as found in nature.
  • vector generally refers to a nucleic acid molecule that can transport another nucleic acid molecule connected thereto.
  • Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques.
  • viral vector in which a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus).
  • virus e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication
  • vectors are integrated into the genome of the host cell after being introduced into the host cell, and are thereby replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors used in recombinant DNA technology are usually in the form of plasmids. Recombinant expression vectors may contain the nucleic acid of the present application in a form suitable for nucleic acid expression in a host cell, which means that these recombinant expression vectors contain the gene.
  • One or more regulatory elements selected for the host cell to be used for expression which are operably linked to the nucleic acid sequence to be expressed.
  • "operably linked” is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory element is generally intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • promoters e.g., promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • IRES internal ribosome entry sites
  • regulatory elements may include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific.
  • tissue of interest such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes).
  • Regulatory elements can also direct expression in a timing-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific.
  • a vector may comprise one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof.
  • pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retrovirus Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter the SV40 promoter
  • dihydrofolate reductase promoter the ⁇ -actin promoter
  • PGK phosphoglycerol kinase
  • regulatory element may also encompass enhancer elements such as WPRE, CMV enhancer, R-U5' fragment in the LTR of HTLV-I, SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).
  • a vector may be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including fusion molecules or enzymes encoded by nucleic acids as described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion molecules or fusion proteins thereof, etc.).
  • Advantageous vectors include lentiviruses and adeno-associated viruses, and vectors of this type may also be selected to target specific types of cells.
  • the term "codon optimization” generally refers to replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, for example, about or more than about 1, 2, 3, 4, 5, 10, 15,20,25,50 or more codons are maintained simultaneously and a nucleic acid sequence is modified to enhance the method for expression in the host cell of interest.Different species show specific preferences for certain codons with specific amino acids.Codon preference (differences in the use of codons between organisms) is often related to the translation efficiency of messenger RNA (mRNA), and the translation efficiency is considered to depend on the properties of the codons translated (among others) and the availability of specific transfer RNA (tRNA) molecules. The advantage of the tRNA selected in the cell generally reflects the codons most frequently used for peptide synthesis.Therefore, genes can be customized to the best gene expression based on codon optimization in a given organism.Codon utilization tables can be easily obtained, for example, in codon usage
  • codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen, Jacobus, PA), which is also available.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in the sequence encoding the Cas enzyme correspond to the most frequently used codons for a particular amino acid.
  • guide RNA is used interchangeably with “guide RNA” and "gRNA”, which generally refers to a group of nucleic acid molecules that facilitate the specific guidance of RNA-guided nucleases or other effector molecules (usually complexed with gRNA molecules) to target sequences.
  • crRNA and tracrRNA usually exist as two independent RNA molecules, constituting gRNA.
  • tracrRNA generally refers to a scaffold-type RNA that can bind to Cas nucleases
  • crRNA also known as CRISPR RNA, generally refers to a nucleotide sequence that is complementary to the targeted target DNA.
  • crRNA and tracrRNA can also be fused into a single strand, in which case gRNA can also be called single-stranded guide RNA (sgRNA), which has become the most common form of gRNA used by those skilled in the art in CRISPR technology, so the terms "sgRNA” and "gRNA” can have the same meaning in this article.
  • sgRNA can be artificially synthesized or prepared from a DNA template in vitro or in vivo. sgRNA can bind to Cas nucleases or target target DNA, and it can guide Cas nucleases to cut DNA sites complementary to gRNA.
  • crRNA generally comprises a spacer sequence that mediates target recognition and a direct repeat sequence (also referred to herein as "Direct repeat” or “DR sequence”) that forms a complex with the CRISPR-Cas effector protein.
  • a direct repeat sequence also referred to herein as "Direct repeat” or "DR sequence”
  • the spacer sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the CRISPR-Cas system complex to specifically bind to the target sequence.
  • the degree of complementarity between the spacer sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Determining the optimal alignment is within the capabilities of a person of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs. programs, such as but not limited to ClustalW, the Smith-Waterman algorithm in Matlab, Bowtie, Geneious, Biopython, and SeqMan.
  • the spacer sequence is at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides in length. In certain embodiments, the spacer sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15, 10 or less nucleotides in length. In certain embodiments, the spacer sequence is 10-30, 15-25, 15-22, 16-24, 19-25, or 19-22 nucleotides in length. In certain preferred embodiments, the spacer sequence is 20 nucleotides in length.
  • the direct repeat sequence is at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 45, at least 50, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, or at least 70 nucleotides in length.
  • the direct repeat sequence is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15, 10 or less nucleotides in length.
  • the direct repeat sequence is 55-70 nucleotides, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleotides, such as 63-64 nucleotides in length.
  • the direct repeat sequence is 15-40 nucleotides, such as 15-25 nucleotides, such as 20-30 nucleotides, such as 22-36 nucleotides, such as 31 nucleotides in length.
  • the term “about” or “approximately” is generally within an acceptable error range for a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, “about” or “approximately” may mean a range of up to 10% or 20% (i.e., ⁇ 10% or ⁇ 20%).
  • selected from generally refers to including selected objects and all combinations thereof.
  • selected from (:) A, B and C is meant to include all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.
  • This example uses stably transfected 293T cells with a blue-green light reporter system to test the activity of the Cas enzyme provided in this application.
  • the reporter system has a continuously expressed CMV promoter, a sequence encoding a blue fluorescent protein, a sequence encoding a green fluorescent protein, and a gRNA targeting sequence inserted in the middle; the gRNA targeting sequence has random N base sequences on both sides, which can be used to screen Cas enzymes with different protospacer adjacent motifs (PAM) preferences.
  • PAM protospacer adjacent motifs
  • the cleavage activity of the Cas enzyme of the present application will be determined by the fluorescence results displayed by the reporter system.
  • the reporter system without cleavage will stably express blue fluorescent protein and emit blue fluorescence, while the green fluorescent protein cannot be expressed due to the presence of a stop codon before the sequence encoding the green fluorescent protein, and the reporter system will not emit green fluorescence. Only after DNA cleavage occurs near the gRNA targeting sequence, and the cell undergoes a frameshift mutation during the repair of the incision, can the green fluorescent protein be expressed while the blue fluorescent protein is stably expressed.
  • the fluorescence results of Figure 1A show that after transfection with the Cas enzyme provided in the present application (SEQ ID NO: 2), the experimental group cells with cleavage activity will produce a cell population that clearly expresses green fluorescence.
  • the green fluorescent cell population is enriched by flow cytometry, the target region is amplified after the genome is extracted, and the PAM motif required for the gRNA binding of the Cas enzyme provided in the present application can be determined by high-throughput sequencing (as shown in Figure 1B).
  • nucleotide sequence encoding a reporter system is shown below (bold is blue and green fluorescent protein sequences, italics are linker sequences, underline is 2A cleavage peptide sequence, bold underline is an exemplary gRNA targeting sequence containing random N bases (SEQ ID NO: 59)):
  • the Cas enzymes (SEQ ID NOs: 2, 47-51 and 54) provided in the present application the corresponding gRNA and the reporter plasmid with the gRNA targeting site were co-transfected into HEK293T cells.
  • nucleotide sequence encoding a reporter system is shown below (bold is a blue and green fluorescent protein sequence, italics is a linker sequence, and underlined is a 2A cleavage peptide sequence; bold underlined is an exemplary gRNA targeting sequence (SEQ ID NO: 60), wherein the TTTG and TGG at both ends of the sequence will be set to different sequences according to the PAM preference of different Cas enzymes):
  • the Cas enzyme provided in this application is expressed and purified by E. coli, and reacted with a plasmid library containing a PAM library together with an in vitro transcribed gRNA (the gRNA targeting sequence is SEQ ID NO: 59, and it has random N base sequences on both sides to determine the PAM preference), and the Cas enzyme with cutting activity will cut the plasmid library to form linearized DNA.
  • the adapter fragment is connected to the library fragment after DNA linearization, and then the vector and the library fragment connected to the adapter are amplified by specific PCR primers, and high-throughput sequencing is performed.
  • gRNA targeting sequence was SEQ ID NO: 89.
  • the Cas enzyme (SEQ ID NOs: 44) provided in this application and the corresponding gRNA were co-transfected into HEK293T cells. Three days after transfection, the transfected positive cells were enriched by flow sorting, and the target region was amplified after the cells were lysed. The amplified PCR fragment was reacted with T7 endonuclease.
  • the different sites of the Cas enzyme (SEQ ID NO:2) provided by the application are mutated into target amino acids (see the table below), and then the detection as described in Example 2 is performed.
  • the DR (direct repeat) regional sequence and Spacer sequence of gRNA are respectively as shown in SEQ ID NO:64 and 60.
  • the result shown in Fig. 5A shows that the activity of m1, m2, m4, m5, m7, m11, and m15 mutants is significantly improved compared to the wild type. After these effective mutations are superimposed, the activity detection of the same method is performed.
  • FIG. 5B shows that the double mutants formed by the superposition of two mutations selected from the table below can further enhance the activity, and the activity of some double mutants is comparable to AsCas12a;
  • Fig. 5C shows that the triple mutants and quadruple mutants formed by further superimposing effective single mutations and double mutations still maintain the original activity, and the activity is comparable to AsCas12a.
  • the amino acid sequence of AsCas12a for comparison is as shown in SEQ ID NO:127.
  • This example optimizes the editing system comprising the Cas enzyme (SEQ ID NO: 57) and gRNA provided in this application.
  • the detection of Cas enzyme activity adopts the reporting system and detection method described in Example 2.
  • the DR (direct repeat) region of the gRNA was changed to 36nt (SEQ ID NO: 87), 31nt (SEQ ID NO: 91), and 22nt (SEQ ID NO: 90) by molecular cloning.
  • the results shown in Figure 6A show that a DR of 31nt length can maintain 100% Cas enzyme activity.
  • the results shown in Figure 6B show that most of the changes have little effect on the activity, while the change of 31.8 can most preferably improve the activity.
  • This embodiment also changes the length of the spacer region (Spacer) used by gRNA for targeting DNA by molecular cloning to improve its binding efficiency, thereby enhancing the activity of the Cas enzyme.
  • Spacer spacer region
  • the Cas enzyme provided in this application can maintain activity.
  • TTTG was mutated into TTTH, TTVG, TVTG and VTTG (H represents A/C/T, V represents A/C/G, and the three plasmids were mixed in equal proportions and transfected) to construct a new PAM reporter system.
  • H represents A/C/T
  • V represents A/C/G
  • the three plasmids were mixed in equal proportions and transfected
  • the Cas enzyme provided in the present application (SEQ ID NO: 57) was inactivated and then fused with rAPOBEC1 and UGI ( FIG. 9A ) to obtain a cytosine base editor based on the Cas enzyme provided in the present application, and co-transfected with the corresponding gRNA (the DR sequence is shown in SEQ ID NO: 91, and the Spacer sequences targeting the EMX1 site and the VEGFA site are shown in SEQ ID NO: 108 and 109, respectively) into HEK293T cells. After 48 hours of transfection, the transfected cells were sorted, the genome was extracted, the target site was amplified, and the base editing efficiency was determined by Sanger sequencing. The results are shown in FIG. 9B-9C .
  • amino acid sequence of the dEpiCas059m46-CBE editor is as follows (SEQ ID NO: 116, italics are NLS, italic bold is rAPOBEC1, bold is dEpiCas059, italics underline is UGI, underline is P2A cleavage peptide, and ⁇ > is mCherry marker; its plasmid sequence is SEQ ID NO: 117):
  • amino acid sequence of the dAsCas12a-CBE editor is as follows (SEQ ID NO: 118, italic NLS, italic bold rAPOBEC1, bold dAsCas12a, italic underline UGI, underline P2A cleavage peptide, and ⁇ > is mCherry marker; its plasmid sequence is SEQ ID NO: 119):
  • the Cas enzyme (SEQ ID NO: 55) provided in this application was inactivated and then fused with 10 ⁇ GCN4 to recruit the fusion peptide of scFV-P65-HSF1, thereby obtaining a gene activation tool based on the Cas enzyme provided in this application.
  • the principle of this tool is based on the fact that GCN4 can spontaneously recognize and bind to scFV, thereby enriching the P65 and HSF1 effectors with transcriptional activation function near the target site of the Cas enzyme, and then activating the gene expression of the target site.
  • the transcriptional activation tool and the gRNA targeting CXCR4 (Spacer sequence is shown in SEQ ID NOs: 120-123, and the four gRNAs were mixed in equal proportions) were co-transfected into HEK293T cells. After 48 hours of transfection, the cells were collected and stained with PE anti-human CXCR4 antibody (BioLegendg, 306506). The fluorescence intensity of the PE channel was detected by flow cytometry to reflect the expression intensity of CXCR4.
  • the average fluorescence intensity of PE in the transfection-positive group was divided by the average fluorescence intensity of PE in the transfection-negative group to obtain the activation efficiency (MFI fold change), which was used to represent the activation intensity of different activation tools (Figure 10), and then represent the DNA binding effect of different tools.
  • amino acid sequence of the scFV-P65-HSF1 fusion peptide is as follows (SEQ ID NO: 124, italics are NLS, italic bold are P65 and HSF1, bold is scFV, italics underline is HA tag, underline is connecting peptide, and ⁇ > is Flag marker):
  • the amino acid sequence of the dEpiCas057-10 ⁇ GCN4 fusion peptide is shown below (SEQ ID NO: 125, NLS in italics, dEpiCas057 in bold, and GCN4 in ⁇ >):
  • the fusion peptide scFV-P65-HSF1 and dEpiCas057-10 ⁇ GCN4 can be expressed together by the plasmid sequence shown in SEQ ID NO:126.
  • the Cas enzyme provided in the present application that loses its cleavage activity through mutation is suitable for application scenarios of base editing and epigenetic modification editing, and is not limited to other application scenarios based on DNA targeting, such as gene activation, gene silencing, chromosome imaging, etc.

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Abstract

涉及生物医药领域，具体地涉及一种新型Cas酶及其系统和应用。

Description

Cas酶及其系统和应用 技术领域
本申请涉及生物医药领域，具体地涉及一种Cas酶及其系统和应用。
背景技术
基因组测序技术和分析方法的最新进展显著加速了对不同领域的生物活动(从原核生物合成途径到人类病理)的遗传基础的理解。为了充分理解和评估基因测序技术所产生的巨量信息，需要在基因组和表观基因组操作技术的规模、效率和易用性等方面进行相应的提高。这些新的基因组和表观基因组工程改造技术将加速许多领域的新应用的发展，包括生物技术、农业和人类治疗学。
成簇规律间隔短回文重复序列(CRISPR)和CRISPR相关联(Cas)基因，统称为CRISPR-Cas或CRISPR/Cas系统，目前已被理解为对细菌和古细菌提供抗噬菌体感染的免疫。原核生物适应性免疫的CRISPR-Cas系统是一组极其多样的蛋白质效应子、非编码元件以及基因座结构，其中一些例子已经被工程改造并适应于产生重要的生物技术。参与宿主防御的系统的组分包括一种或多种能够修饰DNA或RNA的效应子蛋白和负责将这些蛋白活性靶向噬菌体DNA或RNA上特定序列的RNA指导元件，这些指导元件可被重新编程以靶向可替代的DNA或RNA靶。
CRISPR-Cas系统大致可分为两类：1类系统由多个效应子蛋白构成，2类系统由单个效应子蛋白构成，所述单个效应子蛋白与RNA指导物复合以靶向DNA或RNA底物。2类系统的单亚基效应子组成为工程改造和应用转换提供了更简单的组件集，并且迄今为止一直是可编程效应子的重要来源。以CRISPR-Cas9为例的2类CRISPR-Cas系统的表征和工程改造为基因组编辑和其他方面的多样广泛生物技术应用铺平了道路。然而，除了当前的通过其独特的性质实现了新的应用的CRISPR-Cas系统之外，仍然需要开发强有力的基因组工程工具，即用于修饰核酸和多核苷酸(即DNA、RNA或其任何杂合体、衍生物或修饰物)的替代性的可编程效应子和系统。
发明内容
一方面，本申请提供一种分离的Cas酶，所述Cas酶包含SEQ ID NOs:1-58中任一项所示的氨基酸序列或与所述SEQ ID NOs:1-58中任一项所示的氨基酸序列具有至少约80％同一性的序列。例如，所述Cas酶包含与所述SEQ ID NOs:1-58中任一项所示的氨基酸序列具有约80％、约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％、或约100％同一性的序列。例如，本申请提供了包含如下表所示的氨基酸序列的Cas酶：






































在某些实施方式中，所述Cas酶具有能够结合靶DNA链的催化活性结构域和/或切割所述靶DNA链的催化活性结构域。
在某些实施方式中，所述催化活性结构域包含一个或多个氨基酸的改变，从而使得所述Cas酶仅具有结合靶DNA链的活性，或者具有结合靶DNA链的活性和切割所述靶DNA单链的活性。
另一方面，本申请提供一种融合分子，所述融合分子包含本申请所述的Cas酶和一个或多个异源功能结构域。
在某些实施方式中，所述一个或多个异源功能结构域能够调控一种或多种基因产物的表达。
在某些实施方式中，所述一个或多个异源功能结构域直接或间接地融合在所述Cas酶上。
在某些实施方式中，所述一个或多个异源功能结构域选自自解旋酶、核酸酶、解旋酶-核酸酶、DNA甲基转移酶、DNA羟甲基化酶、组蛋白甲基化酶、组蛋白去甲基化酶、组蛋白乙酰转移酶、组蛋白去乙酰化酶、磷酸酶、激酶、转录(共)活化物、转录阻遏物、DNA结合蛋白、DNA结构蛋白、标志物蛋白、报告物蛋白、荧光蛋白、配体结合蛋白、信号肽、亚细胞定位序列、抗体表位和亲和纯化标签。
在某些实施方式中，所述一个或多个异源功能结构域具有以下活性中的一种或多种：甲基酶活性、脱甲基酶活性、脱氨酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、逆转录酶活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。
另一方面，本申请提供一种工程化的、可编程的、非天然存在的CRISPR-Cas系统，所述系统包含本申请所述的Cas酶或本申请所述的融合分子，和一种或多种指导RNA，所述一种或多种指导RNA在细胞中靶向编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述编码一种或多种基因产物的核酸分子的基因座；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
另一方面，本申请提供一种工程化的、非天然存在的载体系统，所述载体系统包含一种或多种载体，所述一种或多种载体包括：a)第一调节元件，所述第一调节元件可操作地连接到一种或多种指导RNA上，所述一种或多种指导RNA能够与编码一种或多种基因产物的核酸分子的基因座中的靶序列杂交，和b)第二调节元件，所述第二调节元件可操作地连接到本申请所述的Cas酶或本申请所述的融合分子上，其中，所述组分a)和所述组分b)位于所述载体系统的相同或不同载体上，且所述指导RNA在细胞中靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述编码一种或多种基因产物的核酸分子的基因座；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
在某些实施方式中，所述一种或多种基因产物的表达被改变。
在某些实施方式中，所述基因产物的表达被降低或者被增多。
在某些实施方式中，所述基因产物是一种蛋白质。
在某些实施方式中，所述细胞是真核细胞。
在某些实施方式中，所述真核细胞是哺乳动物细胞。例如，所述哺乳动物细胞包括但不限于鼠类、猴、人、农畜、体育用动物和宠物的细胞。
在某些实施方式中，所述哺乳动物细胞是人类细胞。
在某些实施方式中，所述Cas酶是经密码子优化的，用以在真核细胞中进行表达。
在某些实施方式中，所述指导RNA包含融合到tracr序列上的指导序列。
在某些实施方式中，所述指导RNA包含直接重复(Direct repeat)序列和间隔(Spacer)序列，其中所述间隔序列与所述指导RNA靶向的核酸分子结合。
在某些实施方式中，所述直接重复序列的长度为10个至70个核苷酸。
在某些实施方式中，所述直接重复序列的长度为31个至36个核苷酸。
在某些实施方式中，所述直接重复序列包含SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列，或者包含与SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列具有至少95％序列同一性的核苷酸序列。
在某些实施方式中，所述间隔序列的长度为16个至24个核苷酸。
在某些实施方式中，所述指导RNA靶向的核酸分子包含能够与所述间隔序列互补配对的核苷酸序列。
在某些实施方式中，所述系统的所述载体或所述Cas酶还包含一个或多个核定位序列(NLS)。
在某些实施方式中，所述系统通过递送系统被引入所述细胞中，所述递送系统选自病毒粒子、脂质体、脂质纳米颗粒、电穿孔、显微注射和缀合。
另一方面，本申请提供改变一种或多种基因产物的表达的方法，所述方法包括向包含和表达编码所述一种或多种基因产物的核酸分子的细胞中引入一种工程化的、非天然存在的CRISPR-Cas系统，所述系统包含本申请所述的Cas酶或本申请所述的融合分子，和一种或多种指导RNA，所述一种或多种指导RNA靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述基因座，由此改变所述一种或多种基因产物的表达；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
另一方面，本申请提供改变一种或多种基因产物的表达的方法，所述方法包括向包含和表达编码所述一种或多种基因产物的核酸分子的细胞中引入一种工程化的、非天然存在的载体系统，所述载体系统包含一种或多种载体，所述一种或多种载体包括：a)第一调节元件，所述第一调节元件可操作地连接到一种或多种指导RNA上，所述一种或多种指导一种或多种指导RNA能够与所述编码一种或多种基因产物的核酸分子的基因座中的靶序列杂交，和b)第二调节元件，所述第二调节元件可操作地连接到本申请所述的Cas酶或本申请所述的融合分子上，其中，所述组分a)和所述组分b)位于所述载体系统的相同或不同载体上，且所述指导RNA在所述细胞中靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述基因座，由此改变所述一种或多种基因产物的表达；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
在某些实施方式中，所述基因产物的表达被降低或者被增多。
在某些实施方式中，所述基因产物是一种蛋白质。
在某些实施方式中，所述细胞是真核细胞。
在某些实施方式中，所述真核细胞是哺乳动物细胞。例如，所述哺乳动物细胞包括但不 限于鼠类、猴、人、农畜、体育用动物和宠物的细胞。
在某些实施方式中，所述哺乳动物细胞是人类细胞。
在某些实施方式中，所述Cas酶是经密码子优化的，用以在真核细胞中进行表达。
在某些实施方式中，所述指导RNA包含融合到tracr序列上的指导序列。
在某些实施方式中，所述指导RNA包含直接重复(Direct repeat)序列和间隔(Spacer)序列，其中所述间隔序列与所述指导RNA靶向的核酸分子结合。
在某些实施方式中，所述直接重复序列的长度为10个至70个核苷酸。
在某些实施方式中，所述直接重复序列的长度为31个至36个核苷酸。
在某些实施方式中，所述直接重复序列包含SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列，或者包含与SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列具有至少95％序列同一性的核苷酸序列。
在某些实施方式中，所述间隔序列的长度为16个至24个核苷酸。
在某些实施方式中，所述指导RNA靶向的核酸分子包含能够与所述间隔序列互补配对的核苷酸序列。
在某些实施方式中，所述系统的所述载体或所述Cas酶还包含一个或多个核定位序列(NLS)。
在某些实施方式中，所述方法包括通过递送系统将所述CRISPR-Cas系统或所述载体系统引入到所述细胞中，所述递送系统选自病毒粒子、脂质体、脂质纳米颗粒、电穿孔、显微注射和缀合。
另一方面，本申请提供编码本申请所述的Cas酶、本申请所述的融合分子或本申请所述的CRISPR-Cas系统的核酸。
另一方面，本申请提供一种细胞，所述细胞包含本申请所述的Cas酶、本申请所述的融合分子、本申请所述的CRISPR-Cas系统、本申请所述的载体系统和/或本申请所述的核酸。
另一方面，本申请提供一种试剂盒，所述试剂盒包含本申请所述的Cas酶、本申请所述的融合分子、本申请所述的CRISPR-Cas系统、本申请所述的载体系统、本申请所述的核酸和/或本申请所述的细胞。
在某些实施方式中，所述试剂盒还包含用于放置所述Cas酶、所述融合分子、所述CRISPR-Cas系统、所述载体系统、所述核酸和/或所述细胞的容器，以及用法说明书。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本 申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A-1B显示的是本申请所述Cas酶在真核细胞内的切割活性的测定流程示意图和荧光结果以及Cas酶的gRNA结合所需要的PAM基序。
图2A-2B显示的是本申请所述Cas酶在真核细胞内的切割活性的测定流程示意图以及不同Cas酶的绿色荧光细胞对比结果。
图3A-3B显示的是本申请所述Cas酶的体外切割活性及其PAM序列的测定流程示意图以及测序结果。
图4显示的是在内源位点检测本申请所述Cas酶活性的位点信息以及扩增检测结果。
图5A-5C显示的是本申请所述Cas酶的不同变体在真核细胞内进行切割活性测定后的GFP荧光结果。
图6A-6B显示的是对本申请所述Cas酶结合不同DR(Direct repeat)区域优化方案处理过的sgRNA在真核细胞内进行切割活性测定后的绿色荧光细胞比例结果。
图7显示的是本申请所述Cas酶结合不同Spacer长度的sgRNA在真核细胞内进行切割活性测定后的绿色荧光细胞比例结果。
图8A-8B显示的是本申请所述Cas酶的不同变体在真核细胞内进行切割活性测定后的绿色荧光细胞比例结果。图8C显示的是本申请所述Cas酶的不同变体在不同PAM报告系统中的切割活性测定结果(绿色荧光细胞比例)。
图9A-9C显示的是包含本申请所述Cas酶的胞嘧啶碱基编辑器的结构示意图，以及其在EMX1和VEGFA两种内源位点上检测碱基编辑效率的结果。
图10显示的是包含本申请所述Cas酶的基因激活表观工具在CXCR4内源位点上检测靶位点基因表达的激活效果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“同一性”可与“同源性”互换地使用，其通常是指两个或多个多肽分子或者两个或多个核酸分子序列之间的关系，该关系通过比较它们的序列确定。在本领域中，“同一性”还指核酸分子或者多肽序列相关性的程度，这可以通过两个或多个核苷酸或两个或多个氨基酸的序列之间的匹配来确定。在本申请中，氨基酸序列的同一性百分比(％)被定义为在比对序列并在必要时引入缺口来达到最大百分比序列同一性，而不将任意保守取代视为序列同一性的部分之后，候选序列中与参考多肽序列中的氨基酸残基相同的氨基酸残基占残基总数的百分比。可以以本领域技术之内的多种方式来达到以测定百分比氨基酸序列同一性为目的的比对，例如，使用公开可得的计算机软件，如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数，包括在所比较的序列的全长内达到最大比对所需的任意算法。在某些实施方式中，多肽分子或核酸分子序列的同一性百分比(％)的计算还可以基于序列突变类型确定残基总数。突变类型包括在序列任一端或两端的插入(延伸)、在序列任一端或两端的缺失(截短)、一个或多个氨基酸/核甘酸的置换/替代、在序列内部的插入、在序列内部的缺失。举多肽的氨基酸序列为例(核苷酸序列同理)，如果突变类型为以下中的一种或多种：一个或多个氨基酸/核苷酸的置换/替代、在序列内部的插入和在序列内部的缺失，则残基总数以比较的分子中较大者来计算。如果突变类型还包括在序列任一端或两端的插入(延伸)或在序列任一端或两端的缺失(截短)或在序列内部的插入和在序列内部的缺失，则在任一端或两端或内部插入或缺失的氨基酸的数量(例如，在两端插入或缺失的数量小于20个)并不计入残基总数中。在计算同一性百分比时，可将正在比较的序列以产生序列之间最大匹配的方式比对，并通过特定算法解决比对中的空位(如果存在的话)。
在本申请中，“催化活性结构域”是指Cas蛋白(酶)内展现显著二级结构含量的可鉴别或可确定的保守结构实体且该保守结构为Cas蛋白(酶)实现例如结合和/或切割多核苷酸功能的区域。示例性的催化活性结构域可以是Cas9家族的蛋白酶，其具有两个催化活性结构域，一个是类HNH的，它的作用是切断与指导RNA配对的单链多核苷酸(目标链)，另一个结构域是类RuvC的，它的作用是切断目标链的互补链。
在本申请中，术语“结合”(例如，关于多肽或蛋白酶的靶DNA结合(催化活性)结构域)通常是指大分子之间(例如，蛋白质与核酸之间)的非共价相互作用。当在非共价相互作用的状态下，大分子被称作“缔合”或“相互作用”或“结合”(例如，当分子X被称作与分子Y相互作用时，意指分子X以非共价方式结合分子Y)。应了解，不是所有的结合相互作用组分都需要为序列特异性的(例如，与DNA骨架中的磷酸酯残基接触)，但结合相互作用的一些部分可为序列特异性的。
在申请中，术语“融合分子”通常是指至少两个部分组成的分子(bipartite molecule)，其包含本申请的酶(蛋白质或肽)，该酶与至少一个其他部分偶联，从而形成单个实体。该酶和该至少一个其他部分可以由接头分开，或者可以直接偶联。该至少一个其他部分可以在N端、C端或末端氨基酸外的任意氨基酸处与本申请的酶融合。其他部分可以与已经包含在该融合分子中的部分融合。本领域的技术人员充分了解用于确定本申请的融合分子中的部分的测定最佳顺序和/或组合。通常，当融合分子包含本申请的酶和至少一种其他肽时，该术语不包括这样的融合分子，在该融合分子中，融合产生天然存在的肽。
在本申请中，术语“异源”通常是指分别不存在于天然核酸或蛋白质中的核苷酸或多肽序列。在本申请的某些实施方式中，术语“异源功能结构域”可指除了本申请所述的Cas酶之外的约或多于约1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个结构域或本申请所述的融合分子的一部分。可以包含在本申请所述的融合分子中或者可以与本申请所述的Cas酶融合的异源功能结构域的实例包括但不限于，表位标签、报告基因序列、以及具有下列活性的一个或多个的蛋白质结构域：甲基酶活性、脱甲基酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。报告基因的实例包括，但不限于，谷胱甘肽-S-转移酶(GST)、辣根过氧化物酶(HRP)、氯霉素乙酰转移酶(CAT)、β-半乳糖苷酶、β-葡糖醛酸糖苷酶、萤光素酶、绿色荧光蛋白(GFP)、HcRed、DsRed、青荧光蛋白(CFP)、黄色荧光蛋白(YFP)、以包括蓝色荧光蛋白(BFP)的自发荧光蛋白。Cas酶可以融合到编码一种蛋白质或蛋白质片段的基因序列上，所述蛋白质或蛋白质片段结合DNA分子或结合其他细胞分子，其包括，但不限于，麦芽糖结合蛋白(MBP)、S-tag、Lex A DNA结合结构域(DBD)融合物、GAL4DNA结合结构域融合物、以及单纯疱疹病毒(HSV)BP16蛋白融合物。可以形成包含Cas酶的融合分子的一部分的另外的结构域描述于US20110059502中，通过引用将其并入本文。
在本申请中，术语“表达”通常是指以此从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后以此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。
在本申请中，术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用。它们通常是指具有任何长度的核苷酸的聚合形式，是脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构，并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。如果存在，可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后，如通过与标记的组分缀合来进一步修饰。
在本申请中，术语“非天然存在的”和“工程化的”可互换地使用。当它们意指核酸分子、多肽、或其组合和其系统时，通常表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
在本申请中，术语“载体”通常是指一种核酸分子，它能够运送与其连接的另一种核酸分子。载体包括但不限于，单链、双链、或部分双链的核酸分子；包括一个或多个自由端、无自由端(例如环状的)的核酸分子；包括DNA、RNA、或两者的核酸分子；以及本领域已知的其他多种多样的多核苷酸。一种类型的载体是“质粒”，其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。在重组DNA技术中使用的普通表达栽体通常是质粒形式。重组的表达载体可包含处于适合于在宿主细胞中的核酸表达的形式的本申请的核酸，这意味着这些重组表达载体包含基 于待用于表达的宿主细胞而选择的一种或多种调节元件，所述调节元件可操作地连接至待表达的核酸序列。在重组表达载体内，“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调节元件(例如，处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。
在本申请中，术语“调节元件”通常旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)。这样的调节序列例如描述于戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥，加利福尼亚州(1990)中。在某些实施方式中，调节元件可包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。调节元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。在某些实施方式中，一个载体可包含一个或多个pol III启动子(例如1、2、3、4、5、或更多个pol III启动子)、一个或多个pol II启动子(例如1、2、3、4、5、或更多个pol II启动子)、一个或多个pol I启动子(例如1、2、3、4、5、或更多个pol I启动子)、或其组合。pol III启动子的实例包括但不限于U6和H1启动子。pol II启动子的实例包括但不限于逆转录劳斯肉瘤病毒(RSV)LTR启动子(任选地具有RSV增强子)、巨细胞病毒(CMV)启动子(任选地具有CMV增强子)、SV40启动子、二氢叶酸还原酶启动子、β-肌动蛋白启动子、磷酸甘油激酶(PGK)启动子、和EF1α启动子。术语“调节元件”还可以涵盖的是增强子元件，如WPRE、CMV增强子、在HTLV-I的LTR中的R-U5’片段、SV40增强子；以及在兔β-珠蛋白的外显子2与3之间的内含子序列(《美国国家科学院院刊》(Proc.Natl.Acad.Sci.USA.)，第78(3)卷，第1527-31页，1981)。
本领域技术人员将理解，表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽，包括由如本申请所述的核酸编码的融合分子或酶(例如，规律间隔成簇短回文重复序列(CRISPR)转录物、蛋白质、酶、其突变体形式、其融合分子或融合蛋白，等等)。有利的载体包括慢病毒以及腺相关病毒，并且也可选择此类型的载体以靶向具体类型的细胞。
在本申请中，术语“密码子优化”通常是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子，例如约或多于约1、2、3、4、5、10、 15、20、25、50个、或更多个密码子同时维持该天然氨基酸序列而修饰一个核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于具有特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于(除其他之外)被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。例如，参见中村Y.(Nakamura Y.)等人，Codon usage tabulated from the international DNA sequence databases:status for the year 2000，《核酸研究》(Nucl.Acids Res.)28:292(2000年)。用于密码子优化特定的序列以便在特定的宿主细胞中表达的计算机算法也是可获得的，如Gene Forge(Aptagen公司，Jacobus，PA)，也是可获得的。在某些实施方式中，在编码Cas酶的序列中的一个或多个密码子(例如1、2、3、4、5、10、15、20、25、50个、或更多个、或所有密码子)相对应于对于特定氨基酸最频繁使用的密码子。
在本申请中，术语“指导RNA”与“guide RNA”、“gRNA”可互换地使用，其通常是指促进将RNA指导的核酸酶或其他效应分子(通常与gRNA分子复合)特异性指导到靶序列上的一组核酸分子。在自然界中，crRNA和tracrRNA通常作为两个独立的RNA分子存在，组成gRNA。术语“tracrRNA”通常是指可与Cas核酸酶结合的支架型RNA，术语“crRNA”也称为CRISPR RNA，通常是指与所靶向的目标DNA互补的一段核苷酸序列。crRNA和tracRNA也可以融合成为单链，此时gRNA也可称为单链指导RNA(sgRNA)，sgRNA已成为本领域技术人员在CRISPR技术中使用的gRNA的最常见的形式，因此术语“sgRNA”和“gRNA”在本文中可具有相同的含义。sgRNA可以人工合成，也可以在体外或体内由DNA模板制备。sgRNA可以结合Cas核酸酶，也可以靶向目标DNA，其可引导Cas核酸酶切割与gRNA互补的DNA位点。
如本申请所使用的，crRNA通常包含介导靶识别的间隔(Spacer)序列和与CRISPR-Cas效应蛋白形成复合物的直接重复序列(本文中也称为“Direct repeat”或“DR序列”)。在某些情况下，间隔序列(也称导向序列)是与靶序列具有足够互补性从而与所述靶序列杂交并引导CRISPR-Cas系统复合物与所述靶序列的特异性结合的任何多核苷酸序列。在某些实施方案中，当最佳比对时，间隔序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程 序，例如但不限于ClustalW、Matlab中的Smith-Waterman算法、Bowtie、Geneious、Biopython以及SeqMan。
在某些实施方案中，所述间隔序列在长度上为至少5个、至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少35个、至少40个、至少45个或至少50个核苷酸。在某些实施方案中，所述间隔序列在长度上为不超过50个、45个、40个、35个、30个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述间隔序列在长度上为10-30个、15-25个、15-22个、16-24个、19-25个、或19-22个核苷酸。在某些优选的实施方案中，所述间隔序列的长度为20个核苷酸。
在某些实施方案中，所述直接重复序列在长度上为至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少31个、至少32个、至少33个、至少34个、至少35个、至少40个、至少45个、至少50个、至少55个、至少56个、至少57个、至少58个、至少59个、至少60个、至少61个、至少62个、至少63个、至少64个、至少65个或至少70个核苷酸。在某些实施方案中，所述直接重复序列在长度上为不超过70个、65个、64个、63个、62个、61个、60个、59个、58个、57个、56个、55个、50个、45个、40个、35个、34个、33个、32个、31个、30个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述直接重复序列在长度上为55-70个核苷酸，例如55-65个核苷酸，例如60-65个核苷酸，例如62-65个核苷酸，例如63-64个核苷酸。在某些实施方案中，所述直接重复序列在长度上为15-40个核苷酸，例如15-25个核苷酸，例如20-30个核苷酸，例如22-36个核苷酸，例如31个核苷酸。
在本申请中，术语“包含”和“包括”可互换地使用，其通常是指包括明确指定的特征，但不排除其他要素。术语“至少”通常是指包含本数的情况。
在本申请中，术语“约”或“大约”通常由本领域普通技术人员确定的针对特定值的可接受误差范围内，其部分取决于怎样测量或确定该值，即，测量系统的限制。例如，“约”或“大约”可意指按照本领域的实践在1或超过1个标准偏差内。或者，“约”或“大约”可意指至多10％或20％的范围(即，±10％或±20％)。
在本申请中，术语“选自”通常是指包括选择的对象以及其所有组合。例如“选自(：)A、 B和C”意指包括A、B和C的所有组合，例如，A、B、C、A+B、A+C、B+C或A+B+C。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的酶及其融合分子、方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
测定本申请的Cas酶在真核细胞内的切割活性
本实施例采用带有蓝-绿光报告系统的稳转293T细胞测试本申请提供的Cas酶的活性。如图1所示，该报告系统带有持续表达的CMV启动子、编码蓝色荧光蛋白的序列、编码绿色荧光蛋白的序列和插入在中间的gRNA靶向序列；gRNA靶向序列的两边带有随机的N碱基序列，可以用来筛选不同的原型间隔区相邻基序(PAM)偏好的Cas酶。
本申请的Cas酶切割活性将通过报告系统显现的荧光结果测定，未发生切割的报告系统将稳定地表达蓝色荧光蛋白从而发出蓝色荧光，而编码绿色荧光蛋白的序列前由于存在终止密码子而使得绿色荧光蛋白不能表达，报告系统因而不会发出绿色荧光。只有在gRNA靶向序列附近发生DNA切割后，细胞在修复切口过程中发生移码突变，才能在蓝色荧光蛋白稳定表达的同时表达出绿色荧光蛋白。图1A的荧光结果表明，在转染了本申请提供的Cas酶(SEQ ID NO:2)后，具有切割活性的实验组细胞会产生明显的表达绿色荧光的细胞群。
将绿色荧光的细胞群用流式分选富集，提取基因组后扩增target区域，通过高通量测序就能确定本申请提供的Cas酶的gRNA结合所需要的PAM基序(如图1B所示)。
在本实施例中，编码报告系统的示例性核苷酸序列如下所示(粗体为蓝色和绿色荧光蛋白序列，斜体为连接序列，下划线为2A剪切肽序列，粗体下划线为示例性的包含随机N碱基的gRNA靶向序列(SEQ ID NO:59))：

实施例2
测定本申请的Cas酶在真核细胞内的切割活性强弱
将本申请提供的Cas酶(SEQ ID NOs:2、47-51和54)、对应的gRNA和带有gRNA靶向位点的报告质粒共转染到HEK293T细胞中，通过改造报告质粒上的靶向序列，可以快速构 建不同Cas酶所需的报告系统。如图2A所示，未发生切割的报告系统细胞只表达蓝色荧光蛋白，绿色荧光蛋白因为被隔断而无法正常表达，此时报告系统仅发出蓝色荧光。在gRNA靶向位点发生切割后，细胞会通过同源重组修复DNA，从而获得完整的绿色荧光蛋白表达框，从而使报告系统发出绿色荧光。
具有更强切割活性的Cas酶会产生更高比例的DNA切割，从而得到更多拷贝的修复后的绿色荧光蛋白，细胞显示出更强的绿色荧光，因此本实施例通过比较绿色荧光细胞的比例和荧光强度，快速比较不同Cas酶的切割活性强弱，其比较结果如图2B所示。
在本实施例中，编码报告系统的示例性核苷酸序列如下所示(粗体为蓝色和绿色荧光蛋白序列，斜体为连接序列，下划线为2A剪切肽序列；粗体下划线为示例性的gRNA靶向序列(SEQ ID NO:60)，其中该序列两端的TTTG和TGG将会根据不同Cas酶的PAM偏好设置成不同序列)：

实施例3
测定本申请的Cas酶的体外切割活性及其PAM序列
如图3A所示的本实施例的实验设计流程图，将本申请提供的Cas酶用大肠杆菌表达并纯化，并与体外转录的gRNA(gRNA靶向序列为SEQ ID NO:59，且其两边带有随机的N碱基序列用以测定PAM偏好)一起与包含PAM文库的质粒文库反应，有切割活性的Cas酶会将质粒文库切断，形成线性化DNA。通过体外连接，将接头片段连接到DNA线性化后的文库片段上，然后通过特异的PCR引物将载体和连接上接头的文库片段扩增，并进行高通量测序。
图3B所示的分析测序结果显示了有切割活性的Cas酶对于PAM基序的偏好性，通过这些结果可表明，EpiCas037(SEQ ID NO:36)、EpiCas044(SEQ ID NO:43)、EpiCas040(SEQ ID NO:39)和EpiCas045四个蛋白(SEQ ID NO:44)都具有切割活性，而且都偏好富含T的 PAM基序。
实施例4
在内源位点检测本申请的Cas酶活性
根据实施例3确定的不同Cas酶的PAM基序，在HEK293T的内源位点找到符合条件的位点，构建gRNA(gRNA靶向序列为SEQ ID NO:89)。将本申请提供的Cas酶(SEQ ID NOs:44)和对应的gRNA共同转染到HEK293T细胞，转染3天后通过流式分选富集转染阳性的细胞，裂解细胞后扩增目的区域。将扩增后的PCR片段和T7内切酶反应，如果Cas酶切割了目的位点，将会形成各种不同的随机修复的序列，这些序列会被T7酶切割产生目的条带以外的条带。因此，图4所示的结果表明该Cas酶在内源位点具有切割活性。
实施例5
测定本申请的Cas酶突变体在真核细胞内的切割活性强弱
通过定点突变，将本申请提供的Cas酶(SEQ ID NO:2)的不同位点突变成目标氨基酸(见下表)，再进行如实施例2所述的检测。其中，gRNA的DR(direct repeat)区域序列和Spacer序列分别如SEQ ID NO:64和60所示。图5A所示的结果表明，m1、m2、m4、m5、m7、m11、m15突变体活性相较于野生型均有显著提升。再将这些有效突变叠加后进行相同方法的活性检测，图5B所示的结果表明，包含由两种选自下表的突变叠加形成的双突变体能进一步提升活性，部分双突变体的活性与AsCas12a相当；图5C所示的结果表明，将有效的单突变和双突变再进一步叠加所形成的三突变体和四突变体仍然保持原有的活性，且活性与AsCas12a相当。用于对照的AsCas12a的氨基酸序列如SEQ ID NO:127所示。

实施例6
CRISPR/Cas编辑系统的优化
本实施例将对包含本申请提供的Cas酶(SEQ ID NO:57)以及gRNA的编辑系统进行优化。其中，对Cas酶活性的检测采用如实施例2所述的报告系统和检测方法。
gRNA优化：
通过分子克隆将gRNA的DR(direct repeat)区域改变成36nt(SEQ ID NO:87)、31nt(SEQ ID NO:91)、22nt(SEQ ID NO:90)，图6A所示的结果表明，31nt长度的DR即能维持100％的Cas酶活性。当通过分子克隆改变DR区域的碱基对以提高其稳定性时(DR为31.1至31.8时，其核苷酸序列如SEQ ID NO:92-99所示)，图6B所示的结果表明，大部分改动对活性影响不大，而31.8的改动能够最优选地提升活性。本实施例还通过分子克隆改变了gRNA用于靶向DNA的间隔区(Spacer)长度，用以提高其结合效率，从而增强Cas酶活性。如图7所示，当Spacer长度从24nt变化至16nt(SEQ ID NO:100-103，60和104-107)，本申请所提供的Cas酶均能维持活性。
Cas酶突变体：
通过定点突变，将本申请提供的Cas酶(SEQ ID NO:57)的不同位点突变成目标氨基酸(见下表)，再进行如实施例2所述的检测。其中，gRNA的DR(direct repeat)区域序列和Spacer序列分别如SEQ ID NO:91和60所示。图8A-8B所示的结果表明，多数单一突变对Cas酶活性的影响不大，如果将其中对活性优化效果较好的单一突变进行叠加，可以发现所形成的双突变体仍能保持较高的活性。
本实施例还在实施例2所述的报告系统基础上，将TTTG分别突变成TTTH、TTVG、TVTG和VTTG(H代表A/C/T，V代表A/C/G，3个质粒等比例混合后转染)，构建出新的PAM报告系统，通过图8C所示的结果发现，部分突变体能够拓宽PAM识别的范围，尤其是在识别VTTG、TTVG的PAM的靶点上，相较于野生型，本申请提供的Cas酶活性获得了显著提升。

实施例7
在内源位点检测失活Cas酶(dCas)效果
dCas-CBE的碱基编辑效果：
将本申请提供的Cas酶(SEQ ID NO:57)进行失活处理，然后与rAPOBEC1、UGI融合(图9A)，从而获得基于本申请提供的Cas酶的胞嘧啶碱基编辑器，并将其与对应的gRNA(DR序列如SEQ ID NO:91所示，靶向EMX1位点和VEGFA位点的Spacer序列分别如SEQ ID NO:108和109所示)共转染到HEK293T细胞中，转染48小时后分选转染阳性的细胞，提取基因组，扩增目的位点，通过Sanger测序确定碱基编辑效率，结果如图9B-9C所示。
其中，dEpiCas059m46-CBE编辑器的氨基酸序列如下所示(SEQ ID NO:116，斜体为NLS，斜粗体为rAPOBEC1，粗体为dEpiCas059，斜体下划线为UGI，下划线为P2A剪切肽，以及<>内为mCherry标记物；其质粒序列为SEQ ID NO:117)：

dAsCas12a-CBE编辑器的氨基酸序列如下所示(SEQ ID NO:118，斜体为NLS，斜粗体为rAPOBEC1，粗体为dAsCas12a，斜体下划线为UGI，下划线为P2A剪切肽，以及<>内为mCherry标记物；其质粒序列为SEQ ID NO:119)：

dCas-SPH的基因激活效果：
将本申请提供的Cas酶(SEQ ID NO:55)进行失活处理，然后与10×GCN4融合，用以招募scFV-P65-HSF1的融合肽，从而获得基于本申请提供的Cas酶的基因激活工具。该工具的原理基于GCN4能够自发与scFV相互识别并结合，进而将具有转录激活功能的P65和HSF1效应物富集到Cas酶的靶位点附近，继而激活靶位点的基因表达。
将转录激活工具和靶向CXCR4的gRNA(Spacer序列如SEQ ID NOs:120-123所示，该4条gRNA被等比例混合)共转染到HEK293T细胞中，转染48小时后，收集细胞，用PE anti-human CXCR4抗体染色(BioLegendg，306506)，通过流式细胞仪检测PE通道荧光强度，用以反映CXCR4的表达强度。将转染阳性群的PE平均荧光强度，除以转染阴性群的PE平均荧光强度，得到激活效率(MFI fold change)，用以代表不同激活工具的激活强度(图10)，进而代表不同工具的DNA结合效果。
其中，scFV-P65-HSF1融合肽的氨基酸序列如下所示(SEQ ID NO:124，斜体为NLS，斜粗体为P65和HSF1，粗体为scFV，斜体下划线为HA标签，下划线为连接肽，以及<>内为Flag标记物)：
dEpiCas057-10×GCN4融合肽的氨基酸序列如下所示(SEQ ID NO:125，斜体为NLS，粗体为dEpiCas057，以及<>内为GCN4)：

融合肽scFV-P65-HSF1和dEpiCas057-10×GCN4可由SEQ ID NO:126所示的质粒序列一同表达。
以上实验证明了通过突变失去切割活性的本申请提供的Cas酶适用于碱基编辑以及表观修饰编辑的应用场景，并且不限于其他基于DNA靶向的应用场景，如基因激活、基因沉默、染色体成像等等。

Claims (47)

  1. 一种分离的Cas酶，所述Cas酶包含SEQ ID NOs:1-58中任一项所示的氨基酸序列或与所述SEQ ID NOs:1-58中任一项所示的氨基酸序列具有至少约80％同一性的序列。
  2. 根据权利要求1所述的Cas酶，所述Cas酶具有能够结合靶DNA链的催化活性结构域和/或切割所述靶DNA链的催化活性结构域。
  3. 根据权利要求1-2中任一项所述的Cas酶，所述催化活性结构域包含一个或多个氨基酸的改变，从而使得所述Cas酶仅具有结合靶DNA链的活性，或者具有结合靶DNA链的活性和切割所述靶DNA单链的活性。
  4. 一种融合分子，所述融合分子包含权利要求1-3中任一项所述的Cas酶和一个或多个异源功能结构域。
  5. 根据权利要求4所述的融合分子，所述一个或多个异源功能结构域能够调控一种或多种基因产物的表达。
  6. 根据权利要求4-5中任一项所述的融合分子，所述一个或多个异源功能结构域直接或间接地融合在所述Cas酶上。
  7. 根据权利要求4-6中任一项所述的融合分子，所述一个或多个异源功能结构域选自自解旋酶、核酸酶、解旋酶-核酸酶、DNA甲基转移酶、DNA羟甲基化酶、组蛋白甲基化酶、组蛋白去甲基化酶、组蛋白乙酰转移酶、组蛋白去乙酰化酶、磷酸酶、激酶、转录(共)活化物、转录阻遏物、DNA结合蛋白、DNA结构蛋白、标志物蛋白、报告物蛋白、荧光蛋白、配体结合蛋白、信号肽、亚细胞定位序列、抗体表位和亲和纯化标签。
  8. 根据权利要求4-7中任一项所述的融合分子，所述一个或多个异源功能结构域具有以下活性中的一种或多种：甲基酶活性、脱甲基酶活性、脱氨酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、逆转录酶活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。
  9. 一种工程化的、可编程的、非天然存在的CRISPR-Cas系统，所述系统包含权利要求1-3中任一项所述的Cas酶或权利要求4-8中任一项所述的融合分子，和一种或多种指导RNA，所述一种或多种指导RNA在细胞中靶向编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述编码一种或多种基因产物的核酸分子的基因座；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
  10. 一种工程化的、非天然存在的载体系统，所述载体系统包含一种或多种载体，所述一种或多种载体包括：
    a)第一调节元件，所述第一调节元件可操作地连接到一种或多种指导RNA上，所述一种或多种指导RNA能够与编码一种或多种基因产物的核酸分子的基因座中的靶序列杂交， 和
    b)第二调节元件，所述第二调节元件可操作地连接到权利要求1-3中任一项所述的Cas酶或权利要求4-8中任一项所述的融合分子上，
    其中，所述组分a)和所述组分b)位于所述载体系统的相同或不同载体上，且所述指导RNA在细胞中靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述编码一种或多种基因产物的核酸分子的基因座；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
  11. 根据权利要求9-10中任一项所述的系统，所述一种或多种基因产物的表达被改变。
  12. 根据权利要求9-11中任一项所述的系统，所述基因产物的表达被降低或者被增多。
  13. 根据权利要求9-12中任一项所述的系统，所述基因产物是一种蛋白质。
  14. 根据权利要求9-13中任一项所述的系统，所述细胞是真核细胞。
  15. 根据权利要求9-14中任一项所述的系统，所述真核细胞是哺乳动物细胞。
  16. 根据权利要求9-15中任一项所述的系统，所述哺乳动物细胞是人类细胞。
  17. 根据权利要求9-16中任一项所述的系统，所述Cas酶是经密码子优化的，用以在真核细胞中进行表达。
  18. 根据权利要求9-17中任一项所述的系统，所述指导RNA包含融合到tracr序列上的指导序列。
  19. 根据权利要求9-18中任一项所述的系统，所述指导RNA包含直接重复(Direct repeat)序列和间隔(Spacer)序列，其中所述间隔序列与所述指导RNA靶向的核酸分子结合。
  20. 根据权利要求19所述的系统，所述直接重复序列的长度为10个至70个核苷酸。
  21. 根据权利要求19或20所述的系统，所述直接重复序列的长度为31个至36个核苷酸。
  22. 根据权利要求19-21中任一项所述的系统，所述直接重复序列包含SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列，或者包含与SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列具有至少95％序列同一性的核苷酸序列。
  23. 根据权利要求19所述的系统，所述间隔序列的长度为16个至24个核苷酸。
  24. 根据权利要求19或23所述的系统，所述指导RNA靶向的核酸分子包含能够与所述间隔序列互补配对的核苷酸序列。
  25. 根据权利要求9-24中任一项所述的系统，所述系统的所述载体或所述Cas酶还包含一个或多个核定位序列(NLS)。
  26. 根据权利要求9-25中任一项所述的系统，所述系统通过递送系统被引入所述细胞中，所述递送系统选自病毒粒子、脂质体、脂质纳米颗粒、电穿孔、显微注射和缀合。
  27. 改变一种或多种基因产物的表达的方法，所述方法包括向包含和表达编码所述一种或多种基因产物的核酸分子的细胞中引入一种工程化的、非天然存在的CRISPR-Cas系统，所述系统包含权利要求1-3中任一项所述的Cas酶或权利要求4-8中任一项所述的融合分子，和一种或多种指导RNA，所述一种或多种指导RNA靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述基因座，由此改变所述一种或多种基因产物的表达；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
  28. 改变一种或多种基因产物的表达的方法，所述方法包括向包含和表达编码所述一种或多种基因产物的核酸分子的细胞中引入一种工程化的、非天然存在的载体系统，所述载体系统包含一种或多种载体，所述一种或多种载体包括：
    a)第一调节元件，所述第一调节元件可操作地连接到一种或多种指导RNA上，所述一种或多种指导一种或多种指导RNA能够与所述编码一种或多种基因产物的核酸分子的基因座中的靶序列杂交，和
    b)第二调节元件，所述第二调节元件可操作地连接到权利要求1-3中任一项所述的Cas酶或权利要求4-8中任一项所述的融合分子上，
    其中，所述组分a)和所述组分b)位于所述载体系统的相同或不同载体上，且所述指导RNA在所述细胞中靶向所述编码一种或多种基因产物的核酸分子的基因座，从而指导所述Cas酶或所述融合分子结合和/或切割所述基因座，由此改变所述一种或多种基因产物的表达；并且，所述Cas酶或所述融合分子与所述指导RNA不共同天然存在。
  29. 根据权利要求27或28所述的方法，所述基因产物的表达被降低或者被增多。
  30. 根据权利要求27-29中任一项所述的方法，所述基因产物是一种蛋白质。
  31. 根据权利要求27-30中任一项所述的方法，所述细胞是真核细胞。
  32. 根据权利要求27-31中任一项所述的方法，所述真核细胞是哺乳动物细胞。
  33. 根据权利要求27-32中任一项所述的方法，所述哺乳动物细胞是人类细胞。
  34. 根据权利要求27-33中任一项所述的方法，所述Cas酶是经密码子优化的，用以在真核细胞中进行表达。
  35. 根据权利要求27-34中任一项所述的方法，所述指导RNA包含融合到tracr序列上的指导序列。
  36. 根据权利要求27-35中任一项所述的方法，所述指导RNA包含直接重复(Direct repeat)序列和间隔(Spacer)序列，其中所述间隔序列与所述指导RNA靶向的核酸分子结合。
  37. 根据权利要求36所述的方法，所述直接重复序列的长度为10个至70个核苷酸。
  38. 根据权利要求36或37所述的方法，所述直接重复序列的长度为31个至36个核苷酸。
  39. 根据权利要求36-38中任一项所述的方法，所述直接重复序列包含SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列，或者包含与SEQ ID NO:63-88和90-99中任一项所示的核苷酸序列具有至少95％序列同一性的核苷酸序列。
  40. 根据权利要求36所述的方法，所述间隔序列的长度为16个至24个核苷酸。
  41. 根据权利要求36或40所述的方法，所述指导RNA靶向的核酸分子包含能够与所述间隔序列互补配对的核苷酸序列。
  42. 根据权利要求27-41中任一项所述的方法，所述系统的所述载体或所述Cas酶还包含一个或多个核定位序列(NLS)。
  43. 根据权利要求27-42中任一项所述的方法，所述方法包括通过递送系统将所述CRISPR-Cas系统或所述载体系统引入到所述细胞中，所述递送系统选自病毒粒子、脂质体、脂质纳米颗粒、电穿孔、显微注射和缀合。
  44. 编码权利要求1-3中任一项所述的Cas酶、权利要求4-8中任一项所述的融合分子或权利要求9和11-26中任一项所述的CRISPR-Cas系统的核酸。
  45. 一种细胞，所述细胞包含权利要求1-3中任一项所述的Cas酶、权利要求4-8中任一项所述的融合分子、权利要求9和11-26中任一项所述的CRISPR-Cas系统、权利要求10-26中任一项所述的载体系统和/或权利要求44所述的核酸。
  46. 一种试剂盒，所述试剂盒包含权利要求1-3中任一项所述的Cas酶、权利要求4-8中任一项所述的融合分子、权利要求9和11-26中任一项所述的CRISPR-Cas系统、权利要求10-26中任一项所述的载体系统、权利要求44所述的核酸和/或权利要求33所述的细胞。
  47. 根据权利要求46所述的试剂盒，所述试剂盒还包含用于放置所述Cas酶、所述融合分子、所述CRISPR-Cas系统、所述载体系统、所述核酸和/或所述细胞的容器，以及用法说明书。
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