WO2024254519A2 - Compositions et procédés pour la modification de gènes humains exprimés par des cellules hépatiques - Google Patents

Compositions et procédés pour la modification de gènes humains exprimés par des cellules hépatiques Download PDF

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WO2024254519A2
WO2024254519A2 PCT/US2024/033100 US2024033100W WO2024254519A2 WO 2024254519 A2 WO2024254519 A2 WO 2024254519A2 US 2024033100 W US2024033100 W US 2024033100W WO 2024254519 A2 WO2024254519 A2 WO 2024254519A2
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nucleic acid
sequence
effector protein
guide nucleic
effector
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WO2024254519A3 (fr
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Sean Chen
Lucas Benjamin HARRINGTON
William Douglass WRIGHT
Aaron DELOUGHERY
Wiputra Jaya HARTONO
Benjamin Julius RAUCH
Stepan TYMOSHENKO
Lauren Kelli UYESAKA
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Mammoth Biosciences Inc
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Mammoth Biosciences Inc
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • C12N15/1137—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 against enzymes
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • target nucleic acids are associated with the ALB gene.
  • target nucleic acids are associated with the PCSK9 gene.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas proteins Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR/Cas systems provide immunity in bacteria and archaea against viruses and plasmids by targeting the nucleic acids of the viruses and plasmids in a sequence-specific manner.
  • Native systems contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets.
  • compositions, systems, and methods comprising the same, and uses thereof.
  • compositions, systems, and methods comprise a guide nucleic acid or use thereof.
  • compositions, systems, and methods disclosed herein may leverage nucleic acid modification activities, such as nucleic acid editing. Editing may comprise: insertion, deletion, substitution, or a combination thereof of one or more nucleotides.
  • modification activities comprise nucleic acid cleavage activity, e.g., cleavage of a phosphodiester bond between two nucleotides.
  • compositions, systems and methods are useful for modifying the nucleotide sequence of a target nucleic acid.
  • compositions, systems and methods are useful for the treatment of a disease or disorder associated with the target nucleic acid.
  • the target nucleic acid comprises a human gene encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) or a portion thereof.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • diseases or disorders associated with PCSK9 are familial hypercholesterolemia and coronary artery disease.
  • the guide nucleic acid comprises a spacer sequence comprising a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4 and TABLE 5.
  • the system further comprises an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences recited in TABLE 1, and wherein the effector protein binds to the guide nucleic acid.
  • the effector protein comprises one or more amino acid substitutions.
  • the one or more amino acid substitutions independently comprise one or more conservative substitutions, one or more non- conservative substitutions, or combinations thereof.
  • the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues.
  • the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • the positively charged amino acid residue is Arg (R).
  • the effector protein comprises at least one nuclear localization signal.
  • the system further comprises an effector partner linked to the effector protein.
  • the effector partner is directly fused to N terminus or C terminus of the effector protein by an amide bond.
  • the at least one nuclear localization signal comprises an amino acid sequence that is identical to any one of nucleotide sequences recited in TABLE 2.
  • the effector protein recognizes a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences recited in TABLE 3.
  • PAM protospacer adjacent motif
  • the spacer sequence comprises a nucleotide sequence that is at least partially complementary to a target sequence of a target nucleic acid, wherein the target sequence comprises at least 17 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 11 or a reverse complement thereof.
  • the spacer sequence comprises a nucleotide sequence that is at least 95% identical to any one of the nucleotide sequences recited in TABLE 4 or TABLE 5.
  • the guide nucleic acid further comprises a repeat sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 6.
  • the guide nucleic acid further comprises an intermediary sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 8. In some embodiments, the guide nucleic acid further comprises a handle sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 9. In some embodiments, the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 261 or a reverse complement thereof. In some embodiments, the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 512 or a reverse complement thereof.
  • the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 514 or a reverse complement thereof.
  • the components of the system as recited in compositions of TABLE 10, comprise a combination of: the effector protein or the nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to identical to SEQ ID NO: 1 or 2, and the guide nucleic acid or the nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises: the spacer sequence that is at least 90% identical to any one of the nucleotide sequences of SEQ ID NO: 87-250 and 262-511, and the repeat sequence that is at least 90% identical to any one of the nucleotide sequences of SEQ ID NO: 251-254 or the handle sequence that is at least 90% identical to any one of the nucleotide sequences of SEQ ID NO: 2
  • the effector protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the effector protein comprises one or more amino acid substitutions independently selected from K58W, I80K, N193K, S209F, A218K, E225K, N286K, M295W, M298L, A306K, Y315M or a combination thereof relative to SEQ ID NO: 1.
  • the effector protein recognizes a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences of SEQ ID NOS: 19-81.
  • PAM protospacer adjacent motif
  • the repeat sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 251.
  • the guide nucleic acid is a crRNA.
  • the guide nucleic acid is a single guide RNA (sgRNA).
  • the handle sequence comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences of SEQ ID NO: 257-259.
  • the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 87-234.
  • the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 262-449. In some embodiments, the spacer sequence is at least 90% identical to any one of the nucleotide sequences of SEQ ID NOS: 235-250 and 450-511. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein comprises one or more amino acid substitutions independently selected from L26R, E157A, E164A, E164L, E166A, E166I, E170A, I471T, I489A, I489S, Y490S, Y490A, F491A, F491S, F491G, D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S, D501R, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A, or a combination thereof relative to SEQ ID NO: 2.
  • the effector protein comprises one or more amino acid substitutions independently selected from T5R, L26K, L26R, A121Q, S198R, S223P, E258K, I471T, S579R, F701R, or a combination thereof relative to SEQ ID NO: 2.
  • the effector protein comprises a substitution of L26R, I471T or a combination thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein recognizes a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences of SEQ ID NOS: 53, 78, 82-86 and 513.
  • PAM protospacer adjacent motif
  • the repeat sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 252-254.
  • the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 235-250.
  • the spacer sequence comprises any one of SEQ ID NOS: 450-511.
  • the guide nucleic acid comprises one or more phosphorothioate (PS) backbone modifications, 2’-fluoro (2’-F) sugar modifications, or 2’-O-Methyl (2’OMe) sugar modifications.
  • the guide nucleic acid comprises PS backbone modification between -3 and -2 positions of the repeat sequence present in the guide nucleic acid, wherein the repeat sequence comprises at least 24 nucleotides.
  • the guide nucleic acid further comprises at least one modification between -16 and -12 positions of the repeat sequence present in the guide nucleic acid.
  • the at least one modification comprises 2’OMe sugar modification at -14 position of the repeat sequence present in the guide nucleic acid.
  • the at least one modification comprises 2’OMe sugar modification at -16 position of the repeat sequence present in the guide nucleic acid.
  • the at least one modification comprises PS backbone modification between -13 and -12 positions of the repeat sequence present in the guide nucleic acid.
  • the at least one modification comprises PS backbone modification between -14 and -13 positions of the repeat sequence present in the guide nucleic acid. In some embodiments, the at least one modification comprises PS backbone modification between -15 and -14 positions of the repeat sequence present in the guide nucleic acid.
  • systems comprising a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 565.
  • the system further comprises an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 573, and wherein the effector protein binds to the guide nucleic acid.
  • the systems described herein further comprises a donor nucleic acid.
  • the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of a functional human protein, wherein the human protein comprises an amino acid sequence that is associated with any of disease recited in TABLE 12.
  • the system further comprises a nucleic acid expression vector, wherein the expression vector comprises at least one of the nucleic acid encoding the effector protein; and the nucleic acid encoding the guide nucleic acid.
  • the nucleic acid expression vector further comprises a nucleotide sequence encoding untranslated regions of any one of the sequences recited in TABLE 20.
  • the nucleic acid expression vector further comprises the donor nucleic acid.
  • the nucleic acid expression vector is a viral vector.
  • the viral vector is an adeno associated viral (AAV) vector.
  • the nucleic acid encoding the effector protein is a messenger RNA (mRNA).
  • the mRNA further comprises one or more of the untranslated regions comprising a nucleotide sequence of any one of the nucleotide sequences recited in TABLE 20.
  • the system further comprises a lipid or a lipid nanoparticle.
  • compositions wherein the compositions comprise one or more components of the systems described herein.
  • compositions wherein the compositions comprise the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein.
  • pharmaceutical compositions wherein the pharmaceutical compositions comprise one or more components of the systems described herein or the compositions described herein, and a pharmaceutical acceptable excipient.
  • compositions wherein the pharmaceutical compositions comprise the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein or the compositions described herein, and a pharmaceutical acceptable excipient.
  • methods of treating a disease associated with a mutation or aberrant expression of a protein in a subject in need thereof comprises administering to the subject one or more components of the systems described herein, the compositions described herein, or the pharmaceutical compositions described herein.
  • the method comprises administering to the subject the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein, the compositions described herein, or the pharmaceutical compositions described herein.
  • the subject has a genetic disorder.
  • the subject has one or more mutations.
  • the one or more mutations comprise a point mutation, a single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • the mutation is associated with one or more of protein expression, protein activity, and protein structural stability.
  • the genetic disorder comprises any one of the diseases recited in TABLE 12.
  • the methods comprise contacting the human gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein.
  • the human gene is a human ALB gene or a human PCSK9 gene.
  • methods of modifying human ALB gene comprise contacting the human ALB gene with one or more components of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein.
  • the methods comprise contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein.
  • methods of modifying human PCSK9 gene comprise contacting the human PCSK9 gene with one or more components of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein.
  • the methods comprise contacting the human PCSK9 gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein.
  • methods of inserting a donor nucleic acid in human ALB gene comprise contacting the human ALB gene with one or more components of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein, wherein the system, the composition and the pharmaceutical composition comprise the donor nucleic acid.
  • the methods comprise contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein of any one of the systems described herein, any one of the compositions described herein, or any one of the pharmaceutical compositions described herein, wherein the system, the composition and the pharmaceutical composition comprise the donor nucleic acid.
  • the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of a functional human protein, wherein the human protein comprises an amino acid sequence that is associated with any of diseases recited in TABLE 12.
  • the methods described herein are performed in a cell. In some embodiments, the method is performed in vivo.
  • cells comprising one or more components of the systems described herein or the compositions described herein. Also provided herein are cells comprising the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein or the compositions described herein. Also provided herein are cells that comprises a target nucleic acid modified by the systems described herein or the compositions described herein. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a stem cell, progenitor cell, induced pluripotent stem cell (iPSC) or a cell derived from an iPSC.
  • iPSC induced pluripotent stem cell
  • FIGs. 1A-1C shows relative bioluminescence observed upon treatment of human primary hepatocytes with a system containing the effector protein CasM.265466 (SEQ ID NO: 1) and gRNA targeting intron 1 of human albumin, as described in Example 7.
  • FIGs. 1A-1C shows relative bioluminescence observed upon treatment of human primary hepatocytes with a system containing the effector protein CasM.265466 (SEQ ID NO: 1) and gRNA targeting intron 1 of human albumin, as described in Example 7.
  • FIG.3 shows relative bioluminescence observed upon treatment of human primary hepatocytes with a system containing the effector protein CasM.265466 (SEQ ID NO: 1) and gRNA targeting intron 1 of human albumin, as described in Example 9.
  • FIG.4 shows relative bioluminescence observed upon treatment of human primary hepatocytes with a system containing the effector protein CasPhi.12 (SEQ ID NO: 2) and gRNA targeting intron 1 of human albumin, as described in Example 10.
  • FIG.5 shows relative bioluminescence observed upon treatment of human primary hepatocytes with a system containing the effector protein CasPhi.12 (SEQ ID NO: 2) and gRNA targeting intron 1 of human albumin, as described in Example 11.
  • FIG.6 shows the editing efficiency (% indels) of CasPhi.12 I471T, delivered by LNP, in mice is comparable with Cas9.
  • FIGS. 7A-7F show various guide modifications that were tested.
  • the modifications include one or more 2’-O-Methyl (2’OMe) sugar modifications, shown as , and one or more phosphorothioate (PS) backbone modifications, shown as ⁇ .
  • FIGS.7A-7B show positions of unbiased modifications that were tested.
  • FIGS. 7C-7F show positions of combinatorial modifications that were tested.
  • FIGS. 8A-8B show the effects of introducing chemical modifications to CasPhi.12 guide RNAs.
  • FIGS. 9A-9C show the effects of introducing chemical modifications to CasPhi.12 guide RNAs.
  • FIG. 10 shows results of luciferase assay that were performed to determine editing efficiency of LNPs comprising three variants of WT CasPhi.12 effector protein (SEQ ID NO: 2) in combination with seven guide nucleic acids.
  • FIG. 11 shows % indel activity that was observed for LNPs comprising three variants of WT CasPhi.12 effector protein (SEQ ID NO: 2) in combination with four guide nucleic acids.
  • FIG. 12 shows results of luciferase assay for CasPhi.12 system using two different UTR combinations, UTR1 and UTR11 (from left to right), the sequences of which are recited in TABLE 20.
  • the system comprised RNA encoding CasPhi.12 effector protein variant having L26R and I471T substitutions. DETAILED DESCRIPTION OF THE INVENTION [0034] It is to be understood that both the foregoing general description and the following detailed description are exemplary, and explanatory only, and are not restrictive of the disclosure.
  • the term, “about,” as used herein in reference to a number or range of numbers, is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
  • the terms, “% identical,” “% identity,” “percent identity,” and grammatical equivalents thereof, as used herein, in the context of an amino acid sequence or nucleotide sequence refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100.
  • ALIGN Myers and Miller, Comput Appl Biosci.1988 Mar;4(1):11-7
  • FASTA Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98
  • gapped BLAST Altschul et al., Nucleic Acids Res.1997 Sep 1;25(17):3389-40
  • BLASTP BLASTN
  • GCG Garnier et al., Nucleic Acids Res.1984 Jan 11;12(1 Pt 1):387-95).
  • % complementary refers to the percent of nucleotides in two nucleotide sequences in said nucleic acid molecules of equal length that can undergo cumulative base pairing at two or more individual corresponding positions in an antiparallel orientation. Accordingly, the terms include nucleic acid sequences that are not completely complementary over their entire length, which indicates that the two or more nucleic acid molecules include one or more mismatches. A “mismatch” is present at any position in the two opposed nucleotides that are not complementary.
  • the % complementary is calculated by dividing the total number of the complementary residues by the total number of the nucleotides in one of the equal length sequences, and multiplying by 100.
  • Complete or total complementarity describes nucleotide sequences in 100% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • Partially complementarity describes nucleotide sequences in which at least 20%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some instances, at least 50%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • At least 70%, 80%, 90% or 95%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • “Noncomplementary” describes nucleotide sequences in which less than 20% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • the term, “percent similarity,” or “% similarity,” as used herein, in the context of an amino acid sequence refers to a value that is calculated by dividing a similarity score by the length of the alignment. The similarity of two amino acid sequences can be calculated by using a BLOSUM62 similarity matrix (Henikoff and Henikoff, Proc.
  • a multilevel consensus sequence (or PROSITE motif sequence) can be used to identify how strongly each domain or motif is conserved.
  • the second and third levels of the multilevel sequence are treated as equivalent to the top level.
  • +1 point is assigned. For example, given the multilevel consensus sequence: RLG and YCK, the test sequence QIQ would receive three points.
  • each combination is scored as: Q-R: +1; Q-Y: +0; I-L: +1; I-C: +0; Q-G: +0; Q-K: +1.
  • the highest score is used when calculating similarity.
  • bind refers to a non- covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptide and a nucleic acid; between a polypeptide/guide nucleic acid complex and a target nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner).
  • Non-limiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific.
  • base editor refers to a polypeptide or fusion protein comprising a base editing activity.
  • the polypeptide with base editing activity may be referred to as an effector partner.
  • the base editor can differ from a naturally occurring base editing enzyme. It is understood that any reference to a base editor herein also refers to a base editing enzyme variant.
  • the base editor is functional when the effector protein is coupled to a guide nucleic acid.
  • the guide nucleic acid imparts sequence specific activity to the base editor.
  • the effector protein may comprise a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein).
  • the base editing enzyme comprises deaminase activity.
  • catalytically inactive effector protein refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid.
  • the catalytic activity that is reduced or eliminated is often a nuclease activity.
  • the naturally-occurring effector protein may be a wildtype protein.
  • the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein.
  • the term, “cis cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex), wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid.
  • cleavage occurs within or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.
  • the term, “codon optimized,” as used herein, refers to a mutation of a nucleotide sequence encoding a polypeptide, such as a nucleotide sequence encoding an effector protein, to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded polypeptide remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized nucleotide sequence encoding an effector protein could be used.
  • the intended host cell were a mouse cell
  • a mouse codon-optimized nucleotide sequence encoding an effector protein could be generated.
  • a eukaryotic cell then a eukaryote codon- optimized nucleotide sequence encoding an effector protein could be generated.
  • a prokaryotic cell then a prokaryote codon-optimized nucleotide sequence encoding an effector protein could be generated. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp/codon.
  • nucleic acid molecule or nucleotide sequence refer to the characteristic of a polynucleotide having nucleotides that can undergo cumulative base pairing with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid in antiparallel orientation. For example, when every nucleotide in a polynucleotide or a specified portion thereof forms a base pair with every nucleotide in an equal length sequence of a reference nucleic acid, that polynucleotide is said to be 100% complementary to the sequence of the reference nucleic acid.
  • the upper (sense) strand sequence is, in general, understood as going in the direction from its 5′- to 3′-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.
  • the reverse sequence is understood as the sequence of the upper strand in the direction from its 3′- to its 5′-end, while the “reverse complement” sequence or the “reverse complementary” sequence is understood as the sequence of the lower strand in the direction of its 5′- to its 3′-end.
  • cleavage assay refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid.
  • the cleavage activity comprises cis cleavage activity.
  • cleave in the context of a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond.
  • the result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single-stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double-stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.
  • a nick hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule
  • single strand break hydrolysis of a single phosphodiester bond on a single-stranded molecule
  • double strand break hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (E); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gln (Q), Ser (S), Thr (T).
  • Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic-hydroxyl; Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Gln (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).
  • CRISPR RNA and “crRNA,” as used herein, refer to a type of guide nucleic acid that is RNA comprising a first sequence that is capable of hybridizing to a target sequence of a target nucleic acid and a second sequence that is capable of interacting with an effector protein either directly (by being bound by an effector protein) or indirectly (e.g., by hybridization with a second nucleic acid molecule that can be bound by an effector).
  • the first sequence and the second sequence are directly connected to each other or by a linker.
  • donor nucleic acid refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.
  • edited target nucleic acid refers to a target nucleic acid, wherein the target nucleic acid has undergone an editing, for example, after contact with an effector protein.
  • the editing is an alteration in the sequence of the target nucleic acid.
  • the edited target nucleic acid comprises an insertion, deletion, or substitution of one or more nucleotides compared to the unedited target nucleic acid.
  • effector protein refers to a protein, polypeptide, or peptide that is capable of interacting with a nucleic acid, such as a guide nucleic acid, to form a complex (e.g., a RNP complex), wherein the complex interacts with a target nucleic acid.
  • effector partner refers to a protein, polypeptide or peptide that can, in combination with an effector protein and guide nucleic acid, impart some function or activity that can be used to effectuate modification(s) of a target nucleic acid described herein and/or change expression of the target nucleic acid or other nucleic acids associated with the target nucleic acid, when used in connection with compositions, systems, and methods described herein.
  • engineered modification refers to a structural change of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence.
  • the engineered modifications of a nucleotide sequence can include chemical modification of one or more nucleobases, or a chemical change to the phosphate backbone, a nucleotide, a nucleobase or a nucleoside.
  • the engineered modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that encodes a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known.
  • the engineered modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased.
  • Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro-transcription, cloning, enzymatic, or chemical cleavage, etc. In some instances, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions within the nucleic acid.
  • the term, “functional domain,” as used herein, refers to a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay. Activities include, but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid modifying, nucleic acid cleaving, protein binding. The absence of the functional domain, including mutations of the functional domain, would abolish or reduce activity. [0063] The term, “functional fragment,” as used herein, refers to a fragment of a protein that retains some function relative to the entire protein.
  • Non-limiting examples of functions are nucleic acid binding, nucleic acid editing, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity.
  • a functional fragment comprises a recognized functional domain, e.g., a catalytic domain.
  • the term, “functional protein,” as used herein, refers to protein that retains at least some if not all activity relative to the wildtype protein.
  • a functional protein can also include a protein having enhanced activity relative to the wildtype protein.
  • Assays are known and available for detecting and quantifying protein activity, e.g., colorimetric and fluorescent assays.
  • a functional protein is a wildtype protein.
  • a functional protein is a functional portion of a wildtype protein.
  • the term, “fused,” as used herein, refers to at least two sequences that are connected together, such as by a covalent bond (e.g., an amide bond or a phosphodiester bond) or by a linker.
  • the covalent bond can be formed by a conjugation (e.g., chemical conjugation or enzymatic conjugation) reaction.
  • fusion protein refers to a protein comprising at least two heterologous polypeptides. In some instances, the fusion protein comprises one or more effector proteins and effector partners.
  • an effector protein and effector partner are not found connected to one another as a native protein or complex that occurs together in nature.
  • genetic disease refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.
  • guide nucleic acid refers to a nucleic acid that, when in a complex with one or more polypeptides described herein (e.g., an RNP complex) can impart sequence selectivity to the complex when the complex interacts with a target nucleic acid.
  • a guide nucleic acid is referred to interchangeably as a guide RNA, however it is understood that guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.
  • guide nucleic acids may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.
  • handle sequence refers to a sequence of nucleotides in a single guide RNA (sgRNA), that is: 1) capable of being non-covalently bound by an effector protein and 2) connects the portion of the sgRNA capable of being non-covalently bound by an effector protein to a nucleotide sequence that is hybridizable to a target nucleic acid.
  • the handle sequence comprises an intermediary sequence, that is capable of being non-covalently bound by an effector protein.
  • the handle sequence further comprises a repeat sequence. In such instances, the intermediary sequence or a combination of the intermediary sequence and the repeat sequence is capable of being non-covalently bound by an effector protein.
  • heterologous refers to at least two different polypeptide sequences that are not found similarly connected to one another in a native nucleic acid or protein.
  • a protein that is heterologous to the effector protein is a protein that is not covalently linked by an amide bond to the effector protein in nature.
  • a protein is heterologous when the protein is not encoded by a species that encodes the effector protein.
  • a guide nucleic acid comprises “heterologous” sequences, which means that it includes a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked by a phosphodiester bond in nature.
  • the first sequence is considered to be heterologous with the second sequence, and, in some instances, the guide nucleic acid is referred to as a heterologous guide nucleic acid.
  • a heterologous system comprises at least one component that is not naturally occurring together with remaining components of the heterologous system.
  • hybridize refers to a nucleotide sequence that is able to noncovalently interact, i.e.
  • Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) for both DNA and RNA.
  • guanine (G) can also base pair with uracil (U).
  • G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
  • a guanine (G) can be considered complementary to both an uracil (U) and to an adenine (A).
  • a G/U base-pair when a G/U base-pair can be made at a given nucleotide position, the position is not considered to be non-complementary, but is instead considered to be complementary. While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, hybridizable, partially hybridizable, or for hybridization to occur. Moreover, in some instances, a nucleotide sequence hybridizes over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).
  • the conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables which are well known in the art.
  • the position of mismatches may become important.
  • the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
  • Any suitable in vitro assay may be utilized to assess whether two sequences “hybridize”.
  • One such assay is a melting point analysis where the greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. The conditions of temperature and ionic strength determine the “stringency” of the hybridization.
  • indel refers to an insertion-deletion or indel mutation, which is a type of genetic mutation that results from the insertion and/or deletion of one or more nucleotide in a target nucleic acid.
  • An indel can vary in length (e.g., 1 to 1,000 nucleotides in length) and be detected by any suitable method, including sequencing.
  • the term, “indel percentage,” as used herein, refers to a percentage of sequencing reads that show at least one nucleotide has been edited from the insertion and/or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides edited. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given effector protein.
  • RNA and mediary sequence in a context of a single nucleic acid system, refers to a nucleotide sequence in a handle sequence, wherein the nucleotide sequence is capable of, at least partially, being non-covalently bound to an effector protein to form a complex (e.g., an RNP complex).
  • An intermediary sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein.
  • in vitro refers to describing something outside an organism.
  • an in vitro system, composition or method takes place in a container for holding laboratory reagents such that it is separated from the biological source from which a material in the container is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • the term “in vivo” is used to describe an event that takes place within an organism.
  • ex vivo is used to describe an event that takes place in a cell that has been obtained from an organism. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • length and linked refer to a nucleic acid (polynucleotide) or polypeptide, expressed as “kilobases” (kb) or “base pairs (bp)”.
  • kb kilobases
  • bp base pairs
  • a length of 1 kb refers to a length of 1000 linked nucleotides
  • a length of 500 bp refers to a length of 500 linked nucleotides.
  • a protein having a length of 500 linked amino acids is simply described as having a length of 500 amino acids.
  • linker refers to a molecule that links a first polypeptide to a second polypeptide (e.g., one or more amino acids) or a first nucleic acid to a second nucleic acid (e.g., one or more nucleotides).
  • the linker that links polynucleotides comprise by a phosphodiester bond.
  • the linker that links polypeptides comprise an amide bond.
  • mutation refers to an alteration that changes an amino acid residue or a nucleotide as described herein. Such an alteration can include, for example, deletions, insertions, and/or substitutions.
  • the mutation can refer to a change in structure of an amino acid residue or nucleotide relative to the starting or reference residue or nucleotide.
  • a mutation of an amino acid residue includes, for example, deletions, insertions and substituting one amino acid residue for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein.
  • a mutation of a nucleotide includes, for example, changing one naturally occurring base for a different naturally occurring base, such as changing an adenine to a thymine or a guanine to a cytosine or an adenine to a cytosine or a guanine to a thymine.
  • a mutation of a nucleotide base results in a structural and/or functional alteration of the encoding peptide, polypeptide or protein by changing the encoded amino acid residue of the peptide, polypeptide or protein.
  • a mutation of a nucleotide base does not result in an alteration of the amino acid sequence or function of encoded peptide, polypeptide or protein, also known as a silent mutation. Methods of mutating an amino acid residue or a nucleotide are well known.
  • the terms, “mutation associated with a disease” and “mutation associated with a genetic disorder,” as used herein, refer to the co-occurrence of a mutation and the phenotype of a disease.
  • the mutation occurs in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation.
  • nickase refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid.
  • nickase activity refers to catalytic activity that results in single stranded nucleic acid cleavage of a double stranded nucleic acid.
  • non-naturally occurring and engineered refer to indicate involvement of the hand of man.
  • nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule.
  • a composition or system described herein refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system.
  • a composition includes an effector protein and a guide nucleic acid that do not naturally occur together.
  • an effector protein or guide nucleic acid that is “natural,” “naturally- occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.
  • nuclease and “endonuclease” as used herein refer to an enzyme which possesses catalytic activity for nucleic acid cleavage.
  • nuclease activity refers to catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).
  • nucleic acid refers to a polymer of nucleotides. In some instances, a nucleic acid comprises ribonucleotides, deoxyribonucleotides, combinations thereof, and modified versions of the same.
  • nucleic acid is single- stranded or double-stranded, unless specified.
  • nucleic acids are double stranded DNA (dsDNA), single stranded (ssDNA), messenger RNA, genomic DNA, cDNA, DNA-RNA hybrids, and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • nucleic acids as described herein comprise one or more mutations, one or more engineered modifications, or both.
  • nucleic acid expression vector refers to a plasmid that can be used to express a nucleic acid of interest.
  • nuclear localization signal refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.
  • nucleotide(s) and nucleoside(s) in the context of a nucleic acid molecule having multiple residues, refer to describing the sugar and base of the residue contained in the nucleic acid molecule.
  • nucleosides as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule.
  • nucleobase(s) or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides.
  • nucleotides, nucleosides, and/or nucleobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement).
  • nucleoside analogs such as modified uridines
  • the sequence 5'- AXG where X is any modified uridine, such as pseudouridine, NI-methyl pseudouridine, or 5- methoxyuridine is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5' -CAU).
  • pharmaceutically acceptable excipient, carrier or diluent refers to any substance formulated alongside the active ingredient of a pharmaceutical composition that allows the active ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • Such a substance can be included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating absorption, reducing viscosity, or enhancing solubility.
  • the selection of appropriate substance can depend upon the route of administration and the dosage form, as well as the active ingredient and other factors. Compositions having such substances can be formulated by suitable methods (see, e.g., Remington, The Science and Practice of Pharmacy 23 rd Ed. Academic Press, 2021). [0090]
  • the terms, “polypeptide” and “protein,” as used herein, refer to a polymeric form of amino acids.
  • a polypeptide includes coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, in some instances, polypeptides as described herein comprise one or more mutations, one or more engineered modifications, or both. It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding an N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell.
  • M N-terminal Methionine
  • V Valine
  • a start codon for the heterologous peptide serves as a start codon for the effector protein as well.
  • the natural start codon encoding an amino acid residue sufficient for initiating translation e.g., Methionine (M) or a Valine (V)
  • M Methionine
  • V Valine
  • primary editing enzyme refers to a protein, polypeptide, or fragment thereof that is capable of catalyzing the editing (insertion, deletion, or base-to-base conversion) of a target nucleotide or nucleotide sequence in a nucleic acid.
  • promoter and “promoter sequence,” as used herein, refer to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3’ direction) coding or non-coding sequence. A transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase, can also be found in a promoter region.
  • Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.
  • various promoters including inducible promoters, are used to drive expression by the various vectors of the present disclosure.
  • PAM protospacer adjacent motif
  • a PAM is required for a complex of an effector protein and a guide nucleic acid (e.g., an RNP complex) to hybridize to and edit the target nucleic acid.
  • the complex does not require a PAM to edit the target nucleic acid.
  • REC domain refers to domain in an ⁇ -helical recognition region or lobe.
  • an effector protein contains at least one REC domain (e.g., REC1, REC2) which generally helps to accommodate and stabilize the guide nucleic acid and target nucleic acid hybrid.
  • regulatory element refers to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., a guide nucleic acid) or a coding sequence (e.g., effector proteins, fusion proteins, and the like) and/or regulate translation of an encoded polypeptide.
  • a non-coding sequence e.g., a guide nucleic acid
  • a coding sequence e.g., effector proteins, fusion proteins, and the like
  • peat sequence refers to a sequence of nucleotides in a guide nucleic acid that is capable of, at least partially, interacting with an effector protein.
  • ribonucleotide protein complex and “RNP” as used herein, refer to a complex of one or more nucleic acids and one or more polypeptides described herein. While the term utilizes “ribonucleotides,” it is understood that the one or more nucleic acids comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), biochemically or chemically modified nucleobases (e.g., one or more engineered modifications described herein), or combinations thereof.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • a combination thereof e.g., RNA with a thymine base
  • biochemically or chemically modified nucleobases e.g., one or more engineered modifications described herein
  • RuvC and RuvC domain refer to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain instances, of processing a pre-crRNA. In some instances, the RuvC domain is located near the C-terminus of the effector protein. In some instances, a single RuvC domain comprises RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC- like” domain.
  • RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (https://www.ebi.ac.uk/interpro/).
  • InterPro https://www.ebi.ac.uk/interpro/.
  • a RuvC-like domain is a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons.
  • single guide nucleic acid refers to a guide nucleic acid, wherein the guide nucleic acid is a single polynucleotide chain having all the required sequence for a functional complex with an effector protein (e.g., being bound by an effector protein, including in some instances activating the effector protein, and hybridizing to a target nucleic acid, without the need for a second nucleic acid molecule).
  • an effector protein e.g., being bound by an effector protein, including in some instances activating the effector protein, and hybridizing to a target nucleic acid, without the need for a second nucleic acid molecule.
  • a sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary sequence, a repeat sequence, a spacer sequence and optionally a linker, or a handle sequence and a spacer sequence).
  • linked guide nucleic acid components e.g., an intermediary sequence, a repeat sequence, a spacer sequence and optionally a linker, or a handle sequence and a spacer sequence.
  • spacer sequence refers to a nucleotide sequence in a guide nucleic acid that is capable of, at least partially, hybridizing to an equal length portion of a sequence (e.g., a target sequence) of a target nucleic acid.
  • subject refers to an animal. In some instances, the subject is a mammal. In some instances, the subject is a human.
  • the subject is diagnosed or at risk for a disease.
  • the term, “sufficiently complementary,” as used herein, refers to a first nucleotide sequence that is partially complementarity to a second nucleotide sequence while still allowing the first nucleotide sequence to hybridize to the second nucleotide sequence with enough affinity to permit a biological activity to occur.
  • a biological activity comprises the formation of a complex between two or more components described herein, such as an effector protein and a guide nucleic acid.
  • a biological activity comprises bringing one or more components described herein into proximity of another component, such as bringing an effector protein- guide nucleic acid complex into proximity of a target nucleic acid.
  • a biological activity also comprises permitting a component described herein to act on another component described herein, such as permitting an effector protein to cleave a target nucleic acid.
  • sequences are said to be sufficiently complementary when at least 85% of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • target nucleic acid refers to a nucleic acid that is selected as the nucleic acid for editing, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein.
  • target nucleic acid comprises RNA, DNA, or a combination thereof.
  • a target nucleic acid is single-stranded (e.g., single- stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).
  • target sequence refers to a nucleotide sequence found within a target nucleic acid. Such a nucleotide sequence can, for example, hybridize to a respective length portion of a guide nucleic acid.
  • transcriptional activator refers to a polypeptide or a fragment thereof that can activate or increase transcription of a target nucleic acid molecule.
  • transcriptional repressor refers to a polypeptide or a fragment thereof that is capable of arresting, preventing, or reducing transcription of a target nucleic acid.
  • transgene refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell.
  • a transgene is meant to include (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced.
  • the cell in which transgene expression occurs can be a target cell, such as a host cell.
  • treatment and “treating,” as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit refers to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease undergoes treatment, even though a diagnosis of this disease has not been made.
  • the term, “variant,” as used herein, refers to a form or version of a protein that differs from the wild-type protein. In some instances, a variant comprises a different function or activity relative to the wild-type protein.
  • compositions, systems, and methods comprising a guide nucleic acid or a nucleic acid encoding the guide nucleic acid.
  • the guide nucleic acid hybridizes to a target nucleic acid, wherein the target nucleic acid comprises an intron 1 of ALB gene, PCSK9 gene, a variant thereof, a portion thereof, a reverse complement thereof, or a combination thereof.
  • compositions, systems, and methods further comprise an effector protein or a nucleic acid encoding the effector protein.
  • Polypeptides described herein may bind and, optionally, cleave nucleic acids in a sequence- specific manner. Polypeptides described herein may also cleave the target nucleic acid within a target sequence or at a position adjacent to the target sequence. In some embodiments, a polypeptide is activated when it binds a certain sequence of a nucleic acid described herein, allowing the polypeptide to cleave a region of a target nucleic acid that is near, but not adjacent to the target sequence.
  • a polypeptide may be an effector protein, such as a CRISPR-associated (Cas) protein, which may bind a guide nucleic acid that imparts activity or sequence selectivity to the polypeptide.
  • An effector protein may also be referred to as a programmable nuclease because the nuclease activity of the protein may be directed to different target nucleic acids by way of revising the guide nucleic acid that the protein binds.
  • compositions, systems, and methods comprising guide nucleic acids comprise a first region or sequence, at least a portion of which interacts with a polypeptide.
  • the first sequence comprises a sequence that is similar or identical to an intermediary nucleic acid sequence, a handle, a repeat sequence, or a combination thereof.
  • the guide nucleic acid does not comprise an intermediary nucleic acid.
  • compositions, systems, and methods comprising guide nucleic acids comprise a second sequence that is at least partially complementary to a target sequence of a target nucleic acid, and which, in some embodiments, is referred to as a spacer sequence.
  • effector proteins disclosed herein binds and/or cleaves nucleic acids, including double stranded RNA (dsRNA), single-stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA).
  • polypeptides disclosed herein provides binding activity, cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, integrase activity, recombinase activity or a combination thereof.
  • the compositions, systems, and methods described herein are non-naturally occurring.
  • compositions, systems, and methods comprise an engineered guide nucleic acid (also referred to herein as a guide nucleic acid) or a use thereof.
  • compositions, systems, and methods comprise an engineered protein or a use thereof.
  • compositions, systems, and methods comprise an isolated polypeptide or a use thereof.
  • compositions, methods, and systems described herein are not found in nature.
  • compositions, methods, and systems described herein comprise at least one non-naturally occurring component.
  • compositions, methods, and systems comprise a guide nucleic acid, wherein the nucleotide sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid.
  • compositions, systems, and methods comprise at least two components that do not naturally occur together.
  • disclosed compositions, systems, and methods comprise a guide nucleic acid comprising a first region, at least a portion of which, interacts with a polypeptide, and a second region that is at least partially complementary to a target sequence in a target nucleic acid, wherein the first region and second region do not naturally occur together and/or are heterologous to each other.
  • compositions, systems, and methods comprise a guide nucleic acid and an effector protein that do not naturally occur together.
  • disclosed compositions, systems, and methods comprise a ribonucleotide-protein (RNP) complex comprising an effector protein and a guide nucleic acid that do not occur together in nature.
  • RNP ribonucleotide-protein
  • an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.
  • the guide nucleic acid comprises a non-natural nucleotide sequence.
  • the non-natural nucleotide sequence is a nucleotide sequence that is not found in nature.
  • the non-natural nucleotide sequence comprises a portion of a naturally- occurring nucleotide sequence, wherein the portion of the naturally-occurring nucleotide sequence is not present in nature absent the remainder of the naturally-occurring nucleotide sequence.
  • the guide nucleic acid comprises two naturally-occurring nucleotide sequences arranged in an order or proximity that is not observed in nature.
  • compositions and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid that do not occur together in nature.
  • compositions and systems comprise at least two components that do not occur together in nature, wherein the at least two components comprise at least one of an effector protein, an effector partner and a guide nucleic acid.
  • guide nucleic acids comprise a first nucleotide sequence and a second nucleotide sequence that do not occur naturally together.
  • a guide nucleic acid comprises a naturally- occurring repeat sequence and a spacer sequence that is complementary to a naturally-occurring eukaryotic nucleotide sequence.
  • the guide nucleic acid comprises a repeat sequence that occurs naturally in an organism and a spacer sequence that does not occur naturally in that organism.
  • a guide nucleic acid comprises a first nucleotide sequence that occurs in a first organism and a second nucleotide sequence that occurs in a second organism, wherein the first organism and the second organism are different.
  • the guide nucleic acid comprises a third nucleotide sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second nucleotide sequences of the guide nucleic acid.
  • the guide nucleic acid comprises two heterologous nucleotide sequences arranged in an order or proximity that is not observed in nature. Therefore, compositions and systems described herein are not naturally occurring.
  • compositions, systems, and methods described herein comprise a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof) that is similar to a naturally occurring polypeptide.
  • the polypeptide lacks a portion of the naturally occurring polypeptide.
  • the polypeptide comprises a mutation relative to the naturally-occurring polypeptide, wherein the mutation is not found in nature.
  • the polypeptide also comprises at least one additional amino acid relative to the naturally-occurring polypeptide.
  • the polypeptide comprises a heterologous polypeptide.
  • the polypeptide comprises an addition of at least one nuclear localization signal relative to the natural occurring polypeptide.
  • the polypeptide comprises an addition of a nuclear localization signal relative to the natural occurring polypeptide.
  • a nucleotide sequence encoding the polypeptide is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.
  • compositions, systems and methods comprising a polypeptide or polypeptide system, wherein the polypeptide or polypeptide system described herein comprises one or more effector proteins or variants thereof, one or more effector partners or variants thereof, one or more linkers for peptides, or combinations thereof.
  • Effector Proteins [0122] Provided herein are compositions, systems, and methods comprising an effector protein or a use thereof.
  • an effector protein provided herein interacts with a guide nucleic acid to form a complex.
  • the complex interacts with a target nucleic acid.
  • an interaction between the complex and a target nucleic acid comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid by the effector protein, or combinations thereof.
  • recognition of a PAM sequence within a target nucleic acid may is direct the modification activity of an effector protein.
  • recognition of a PAM sequence adjacent to a target sequence of a target nucleic acid may directs the modification activity of an effector protein.
  • Modification activity of an effector protein or an engineered protein described herein comprises cleavage activity, binding activity, insertion activity, substitution activity, and the like.
  • modification activity of an effector protein results in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or any combination thereof.
  • modification of a target nucleic acid comprises introducing or removing epigenetic modification(s).
  • an ability of an effector protein to edit a target nucleic acid depends upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or combinations thereof.
  • a target nucleic acid comprises a target strand and a non-target strand. Accordingly, in some embodiments, the effector protein edits a target strand and/or a non-target strand of a target nucleic acid.
  • the modification of a target nucleic acid generated by an effector protein results in modulation of the expression of the target nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g., inactivation of a protein binding to an RNA molecule or hybridization).
  • modulation of the expression of the target nucleic acid e.g., increasing or decreasing expression of the nucleic acid
  • modulation of the activity of a translation product of the target nucleic acid e.g., inactivation of a protein binding to an RNA molecule or hybridization.
  • effector proteins disclosed herein provide nucleic acid cleavage activity. In some embodiments, effector proteins provide nuclease activity. In some embodiments, effector proteins provide nickase activity. In general, effector proteins described herein edit a target nucleic acid by cis cleavage activity on the target nucleic acid. In some embodiments, effector proteins disclosed herein comprise a RuvC domain comprising cleavage activity. Effector proteins disclosed herein cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single- stranded DNA (ssDNA).
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • ssDNA single- stranded DNA
  • effector proteins disclosed herein provide catalytic activity (e.g., cleavage activity, nickase activity, nuclease activity, other activity or combinations thereof) similar to that of a naturally-occurring effector protein, such as, for example, a naturally-occurring effector protein with reduced cleavage activity including cis cleavage activity.
  • effector proteins disclosed herein are fused to effector partners or fusion proteins, wherein the effector partners or fusion proteins impart some function or activity not provided by an effector protein.
  • an effector protein comprises a CRISPR-associated (“Cas”) protein.
  • an effector protein functions as a single protein, including a single protein that binds to a guide nucleic acid and editing a target nucleic acid.
  • an effector protein functions as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer).
  • an effector protein when functioning in a multiprotein complex, comprises only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex comprising the other functional activity (e.g., editing a target nucleic acid).
  • an effector protein when functioning in a multiprotein complex, comprises differing and/or complementary functional activity to other effector proteins in the multiprotein complex. Multimeric complexes, and functions thereof, are described in further detail below.
  • an effector protein comprises a modified effector protein having increased modification activity and/or increased substrate binding activity (e.g., substrate selectivity, specificity, and/or affinity).
  • an effector protein comprises a catalytically inactive effector protein having reduced modification activity or no modification activity.
  • an effector protein, or a recombinant nucleic acid encoding an effector protein comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences set forth in TABLE 1.
  • the recombinant nucleic acid encoding the effector protein is operably linked to a nucleotide sequence encoding an untranslated region (UTR), such as a 5’ UTR or a 3’ UTR.
  • UTR untranslated region
  • the recombinant nucleic acid encoding the effector protein is operably linked to a promoter, wherein the promoter is functional in a eukaryotic cell or a prokaryotic cell.
  • the promoter is any one or more of: a constitutive promoter, an inducible promoter, a cell type-specific promoter, and a tissue-specific promoter.
  • the recombinant nucleic acid described herein wherein the promoter is functional in any one of: a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, and a human cell.
  • the recombinant nucleic acid is a nucleic acid expression vector as described herein.
  • compositions, systems and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the amino acid sequence of the effector protein comprises at least 200 contiguous amino acids or more of any one of the amino acid sequences recited in TABLE 1.
  • the amino acid sequence of an effector protein provided herein comprises at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, at least 380, at least 400 contiguous amino acids, at least 420 contiguous amino acids, at least 440 contiguous amino acids, at least 460 contiguous amino acids, at least 480 contiguous amino acids, at least 500 contiguous amino acids, at least 520 contiguous amino acids, at least 540 contiguous amino acids, at least 560 contiguous amino acids, at least 580 contiguous amino acids, at least 600 contiguous amino acids, at least 620 contiguous amino acids, at least 640 contiguous amino acids, at least 660 contiguous amino acids, at least 680 contiguous amino acids, at least 700 contiguous amino acids of any one of the amino acid sequences of TABLE 1.
  • compositions, systems and methods described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1.
  • the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1, wherein the portion does not comprise at least the first 10 amino acids, at least the first 20 amino acids, at least the first 40 amino acids, at least the first 60 amino acids, at least the first 80 amino acids, at least the first 100 amino acids, at least the first 120 amino acids, at least the first 140 amino acids, at least the first 160 amino acids, at least the first 180 amino acids, or at least the first 200 amino acids of any one of the amino acid sequences recited in TABLE 1.
  • the effector protein comprises a portion of any one of the amino acid sequences recited in TABLE 1, wherein the portion does not comprise the last 10 amino acids, the last 20 amino acids, the last 40 amino acids, the last 60 amino acids, the last 80 amino acids, the last 100 amino acids, the last 120 amino acids, the last 140 amino acids, the last 160 amino acids, the last 180 amino acids, or the last 200 amino acids of any one of the amino acid sequences recited in TABLE 1.
  • compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 65% identical to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 70% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 75% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 80% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 97% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% identical to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 99% identical to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is identical to any one of the amino acid sequences as set forth in TABLE 1. [0132] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 80% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 85% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 90% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 95% similar to any one of the amino acid sequences as set forth in TABLE 1.
  • an effector protein provided herein comprises an amino acid sequence that is at least 97% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% similar to any one of the amino acid sequences as set forth in TABLE 1. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is 100% similar to any one of the amino acid sequences as set forth in TABLE 1.
  • compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more alterations comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more amino acid alterations relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more alterations comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty amino acid alterations relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more alterations comprises one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid alterations relative to any one of the amino acid sequences recited in TABLE 1.
  • the effector protein comprising one or more amino acid alterations is a variant of an effector protein described herein.
  • any reference to an effector protein herein also refers to an effector protein variant as described herein.
  • the one or more amino acid alterations comprises conservative substitutions, non- conservative substitutions, deletions, or combinations thereof.
  • an effector protein or a nucleic acid encoding the effector protein comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations or more relative to any one of the amino acid sequences recited in TABLE 1.
  • TABLE 1.1 recites an exemplary sequence of an effector protein described herein.
  • compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more substitutions comprise one or more conservative substitutions, one or more non-conservative substitutions, or combinations thereof.
  • a conservative substitution of a basic amino acid of any one of the amino acid sequences recited in TABLE 1 is a substitution by another basic (positively charged) amino acid (e.g., Lys (K), Arg (R), or His (H)).
  • a non-conservative substitution of acidic (negatively charged) amino acid of any one of the amino acid sequences recited in TABLE 1 is a substitution by a basic (positively charged) amino acid (e.g., Lys (K), Arg (R), or His (H)).
  • compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more conservative substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more conservative substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more conservative substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more non-conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more non-conservative substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more non-conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more non-conservative substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty non-conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • the one or more non-conservative substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more non-conservative substitutions relative to any one of the amino acid sequences recited in TABLE 1.
  • an effector protein disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to any one of the amino acid sequences recited in TABLE 1, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the amino acid sequence in TABLE 1 are conservative amino acid substitutions.
  • an effector protein disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to any one of the amino acid sequences recited in TABLE 1, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the amino acid sequence in TABLE 1 are non-conservative amino acid substitutions.
  • an effector protein disclosed herein comprises an amino acid sequence that is identical to any one of the amino acid sequences recited in TABLE 1 with the exception of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid alterations.
  • an effector protein disclosed herein comprises an amino acid sequence that is identical to any one of the amino acid sequences recited in TABLE 1 with the exception of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-conservative amino acid alterations.
  • an effector protein disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% similar to any one of the amino acid sequences recited in TABLE 1, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the amino acid sequence in TABLE 1 are conservative amino acid substitutions.
  • an effector protein disclosed herein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% similar to any one of the amino acid sequences recited in TABLE 1, wherein all but 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids alterations relative to the amino acid sequence in TABLE 1 are non-conservative amino acid substitutions.
  • an effector protein disclosed herein comprises an amino acid sequence that is similar to any one of the amino acid sequences recited in TABLE 1 with the exception of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid alterations.
  • an effector protein disclosed herein comprises an amino acid sequence that is similar to any one of the amino acid sequences recited in TABLE 1 with the exception of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-conservative amino acid alterations.
  • the one or more amino acid alterations result in a change in activity of the effector protein relative to a naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)).
  • a WT effector protein e.g., SEQ ID NO: 1 or 2
  • the one or more amino acid alterations increase or decrease catalytic activity of the effector protein relative to a naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)).
  • the one or more amino acid alterations result in a catalytically inactive effector protein variant.
  • the effector proteins comprising the one or more amino acid alterations can carry out similar enzymatic reactions as the naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)).
  • a WT effector protein e.g., SEQ ID NO: 1 or 2
  • the one or more amino acid alterations increase or decrease biding activity of the effector protein relative to a naturally-occurring counterpart (a WT effector protein (e.g., SEQ ID NO: 1 or 2)).
  • the variants of the effector protein as described herein can include alterations that provide a beneficial characteristic to effector proteins described herein, including but not limited to, increased activity (e.g., indel activity, catalytic activity, specificity or selectivity and/or affinity for a substrate, such as a target nucleic acid and/or a guide nucleic acid).
  • variants of effector proteins described herein can exhibit an activity that is at least the same or higher than the WT effector protein (e.g., SEQ ID NO: 1 or 2), that is, it has one or more activities that are the same or higher than the effector protein (e.g., SEQ ID NO: 1 or 2) without the variant at the same amino acid position(s).
  • variants can have one or more activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% higher over a WT effector protein (e.g., SEQ ID NO: 1 or 2).
  • WT effector protein e.g., SEQ ID NO: 1 or 2
  • activity of effector proteins described herein or variants thereof can be measured relative to a WT effector protein (e.g., SEQ ID NO: 1 or 2) in a cleavage assay.
  • the effector proteins described herein comprises a substitution of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids with positively charged amino acids. In some embodiments, the effector proteins described herein comprises a substitution of one, two, three, four, five, six, seven, eight, nine, or ten amino acids with positively charged amino acids.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, wherein the effector protein comprises one or more amino acid alterations independently at the positions selected from K58, I80, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, or a combination thereof.
  • the effector protein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% similar to SEQ ID NO: 1, and wherein the effector protein also comprises one or more alterations independently at the positions selected from K58, I80, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, or a combination thereof.
  • the one or more amino acid alterations comprise one or more substitutions independently selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more amino acid alterations comprise one or more substitutions independently selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more amino acid alterations comprise one or more substitutions independently selected from I80K, T84K, C202K, G210K, A218K, D220K, E225K, C246K, Q360K, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more amino acid alterations comprise one or more substitutions independently selected from I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more amino acid alterations comprise one or more substitutions independently selected from K58W, I80R, T84R, K105R, N193K, C202R, S209F, G210R, A218K, A218R, D220R, E225K, E225R, C246R, N286K, M295W, M298L, A306K, Y315M, Q360R, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more alterations comprise one or more substitutions independently selected from K58W, I80K, N193K, S209F, A218R, E225K, N286K, M295W, M298L, A306K, Y315M, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the one or more alterations comprise one or more substitutions independently selected from D237A, D418A, D418N, E335A, and E335Q, or a combination thereof relative to the amino acid sequence of SEQ ID NO: 1.
  • the effector proteins described herein comprises a substitution of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids with positively charged amino acids. In some embodiments, the effector proteins described herein comprises a substitution of one, two, three, four, five, six, seven, eight, nine, or ten amino acids with positively charged amino acids.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, wherein the effector protein comprises one or more amino acid alterations independently at the positions selected from I2, T5, K15, R18, H20, S21, L26, N30, E33, E34, A35, K37, K38, R41, N43, Q54, Q79R, K92E, K99R, S108, E109, H110, G111, D113, T114, P116, K118, E119, A121, N132, K135, Q138, V139, L149, E157, E164, E166, E170, Y180, L182, Q183, K184, S186, K189, S196, S198, K200, I203, S205, K206, Y207, H208, N209, Y
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% similar to SEQ ID NO: 2, wherein the effector protein comprises one or more amino acid alterations independently at the positions selected from I2, T5, K15, R18, H20, S21, L26, N30, E33, E34, A35, K37, K38, R41, N43, Q54, Q79R, K92E, K99R, S108, E109, H110, G111, D113, T114, P116, K118, E119, A121, N132, K135, Q138, V139, L149, E157, E164, E166, E170, Y180, L182, Q183, K184, S186, K189, S196, S198, K200, I203, S205, K206, Y207, H208, N209, Y
  • the one or more amino acid alterations comprise one or more substitutions independently selected from I2R, T5R, K15R, R18R, H20R, S21R, L26R, L26K, N30R, E33R, E34R, A35R, K37R, K38R, R41R, N43R, Q54R, Q79R, K92E, K99R, S108R, E109R, H110R, G111R, D113R, T114R, P116R, K118R, E119S, A121Q, N132R, K135R, Q138R, V139R, L149R, Y180R, L182R, Q183R, K184R, S186R, K189R, K189P, S196R, S198R, K200R, I203R, S205R, K206R, Y207R, H208R, N209R, Y220S, S223P, E258K, K281R, K348
  • the one or more amino acid alterations comprise one or more substitutions independently selected from L26R, E157A, E164A, E164L, E166A, E166I, E170A, I471T, I489A, I489S, Y490S, Y490A, F491A, F491S, F491G, D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S, D501R, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A, or any combinations thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • the one or more amino acid alterations comprise T5R, L26R, L26K, A121Q, S198R, S223P, E258K, I471T, S579R, F701R, or any combinations thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • the one or more amino acid alterations comprise D369A, D369N, D658A, D658N, E567A, E567Q, or any combinations thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • the one or more amino acid alterations comprise E567A or E567Q relative to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, wherein the effector protein comprises an L26R substitution relative to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, wherein the effector protein comprises an I471T substitution relative to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, wherein the effector protein comprises an L26R and I471T substitutions relative to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the effector protein comprises a substitution of L26R, I471T or a combination thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • effector proteins provided herein are a variant of a WT effector protein, wherein the WT effector protein has an amino acid sequence of any one of the amino acid sequences set forth in TABLE 1, and the effector protein comprises one or more amino acid alterations in one or more regions that interact with a substrate, such as a target nucleic acid, an engineered guide nucleic acid, or a guide nucleic acid-target nucleic acid heteroduplex.
  • a substrate such as a target nucleic acid, an engineered guide nucleic acid, or a guide nucleic acid-target nucleic acid heteroduplex.
  • effector proteins provided herein are variants of a WT effector protein, wherein the WT effector protein has an amino acid sequence of any one of the amino acid sequences set forth in TABLE 1, and the effector protein comprises one or more amino acid alterations in a region of the effector protein that comprises a substrate binding activity, a catalytic activity, and/or a binding affinity for a substrate, such as a target nucleic acid, an engineered guide nucleic acid, or a guide nucleic acid-target nucleic acid heteroduplex.
  • effector proteins provided herein are a variant of a reference effector protein, wherein the WT effector protein comprises an amino acid sequence of any one of the amino acid sequences set forth in TABLE 1, and the effector protein comprises one or more amino acid alterations in a RuvC domain, a REC domain, TPID, NTPID, or a combination thereof.
  • compositions, systems and methods described herein comprise a nucleic acid encoding the effector protein, wherein the nucleic acid encoding the effector protein is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • compositions, systems and methods described herein comprise a nucleic acid encoding an effector protein, and one or more UTRs, wherein the effector protein is operably linked to the one or more UTRs, and wherein the nucleic acid is an mRNA.
  • the one or more UTRs comprise a 5’ UTR, a 3’ UTR or a combination thereof.
  • the mRNA encoding the 5’ UTR comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 796 or SEQ ID NO: 798.
  • the mRNA encoding the 3’ UTR comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 797 or SEQ ID NO: 799.
  • compositions, systems and methods described herein comprise an mRNA encoding a 5’ UTR, an effector protein, and 3’ UTR, wherein the 5’ UTR and 3’ UTR are operably linked to the effector protein.
  • the mRNA encoding the 5’ UTR region comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 796
  • the mRNA encoding the 3’ UTR region comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 797.
  • the mRNA encoding the 5’ UTR region comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 798
  • the mRNA encoding the 3’ UTR region comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 799.
  • Effector Partners Provided herein are compositions, systems, and methods comprising one or more effector partners or uses thereof.
  • the effector partner is a heterologous protein an effector protein described herein. In some embodiments, the effector partner is not an effector protein as described herein. In some embodiments, the effector partner imparts a function or activity that is not provided by an effector protein as described herein. In some embodiments, the effector partner comprises a second effector protein or a multimeric form thereof.
  • an effector partner imparts a function or activity to a fusion protein comprising an effector protein that is not provided by the effector protein, including but not limited to nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, dimer forming activity (e.g., pyrimidine dimer forming activity), integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOyl
  • the effector partner is fused or linked to an effector protein described herein. In some embodiments, the effector partner is not fused or linked to the effector protein. In some embodiments, the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein directly or by a linker. In some embodiments, the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein directly or by a linker. In some embodiments, the effector partner is functional when the effector protein is coupled to a guide nucleic acid. In some embodiments, the effector partner is functional when the effector protein is coupled to a target nucleic acid.
  • the guide nucleic acid imparts sequence specific activity to the effector partner.
  • the effector protein comprises a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein) when fused or linked to an effector partner.
  • the effector partner directly or indirectly edits a target nucleic acid. Edits can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid.
  • the effector partner interacts with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid.
  • the effector partner modifies proteins associated with a target nucleic acid.
  • an effector partner modulates transcription (e.g., inhibits transcription, increases transcription) of a target nucleic acid.
  • an effector partner directly or indirectly inhibits, reduces, activates or increases expression of a target nucleic acid.
  • Multimeric Complex Formation Modification Activity [0150]
  • an effector partner inhibits the formation of a multimeric complex of an effector protein.
  • the effector partner promotes the formation of a multimeric complex of the effector protein.
  • RT Reverse Transcriptase
  • systems and methods comprise components or uses of an RT editing system to modify a target nucleic acid.
  • RT editing is also referred to as prime editing or precise nucleobase editing.
  • an RT editing system comprises an effector protein and an effector partner comprising an RT editing enzyme.
  • an RT editing enzyme comprises a polymerase.
  • an RT editing enzyme comprises a reverse transcriptase.
  • a non- limiting example of a reverse transcriptase is an M-MLV RT enzyme and variants thereof having polymerase activity.
  • the M-MLV RT enzyme comprises at least one mutation selected from D200N, L603W, T330P, T306K, and W313F relative to wildtype M-MLV RT enzyme.
  • systems and methods comprise an RT editing enzyme, wherein the RT editing enzyme is not fused or linked to the effector protein.
  • the RT editing enzyme comprises a recruiting moiety that recruits the RT editing enzyme to the target nucleic acid.
  • the RT editing enzyme comprises a peptide that binds an aptamer, wherein the aptamer is located on a guide RNA, template RNA, or combination thereof.
  • the RT editing enzyme is linked to a protein that binds to (or is bound by) the effector protein or a protein linked/fused to the effector protein.
  • an RT editing enzyme requires an RT editing guide RNA (pegRNA) to catalyze editing.
  • the pegRNA identifies a target nucleotide or target sequence in a target nucleic acid to be edited and encoding a new genetic information that replaces the target nucleotide or target sequence in the target nucleic acid.
  • an RT editing enzyme requires a pegRNA and a guide RNA, such as a single guide RNA, to catalyze the editing.
  • the RT editing system comprises a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule except for at least one nucleotide.
  • the template RNA is covalently linked to a guide RNA.
  • the template RNA is not covalently linked to a guide RNA.
  • at least a portion of the template RNA hybridizes to the target nucleic acid.
  • the target nucleic acid is a dsDNA molecule.
  • the pegRNA comprises: a guide RNA comprising a second region that is bound by the effector protein, and a first region comprising a spacer sequence that is complementary to a target sequence of the dsDNA molecule; and a template RNA comprising a primer binding sequence that hybridizes to a primer sequence of the dsDNA molecule that is formed when target nucleic acid is cleaved, and a template sequence that is complementary to at least a portion of the target sequence of the dsDNA molecule with the exception of at least one nucleotide.
  • the at least one nucleotide is incorporated into the target nucleic acid by activity of the RT editing enzyme, thereby modifying the target nucleic acid.
  • the spacer sequence is complementary to the target sequence on a target strand of the dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on a non-target strand of the dsDNA molecule.
  • the primer binding sequence hybridizes to a primer sequence on the non-target strand of the dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the dsDNA molecule. In some embodiments, the target strand is cleaved.
  • the non-target strand is cleaved.
  • Nucleic Acid Modification Activity [0152]
  • effector partners have enzymatic activity that modifies a nucleic acid, such as a target nucleic acid.
  • the target nucleic acid comprises or consists of a ssRNA, dsRNA, ssDNA, or a dsDNA.
  • nuclease activity which comprises the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids, such as that provided by a restriction enzyme, or a nuclease (e.g., FokI nuclease); methyltransferase activity such as that provided by a methyltransferase (e.g., HhaI DNA m5c- methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven
  • effector partners target a ssRNA, dsRNA, ssDNA, or a dsDNA.
  • effector partners target ssRNA.
  • Non-limiting examples of effector partners for targeting ssRNA include, but are not limited to, splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; and RNA-binding proteins.
  • splicing factors e.g., RS domains
  • protein translation components e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G
  • an effector partner comprises an entire protein, or a fragment of the protein (e.g., a functional domain).
  • the functional domain binds or interacts with a nucleic acid, such as ssRNA, including intramolecular and/or intermolecular secondary structures thereof (e.g., hairpins, stem-loops, etc.).
  • the functional domain interacts transiently or irreversibly, directly, or indirectly.
  • a functional domain comprises a region of one or more amino acids in a protein that is required for an activity of the protein, or the full extent of that activity, as measured in an in vitro assay.
  • Activities include but are not limited to nucleic acid binding, nucleic acid editing, nucleic acid mutating, nucleic acid modifying, nucleic acid cleaving, protein binding or combinations thereof.
  • effector partners comprise a protein or domain thereof selected from: endonucleases (e.g., RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N- terminus); SMG5 and SMG6; domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFIIm); exonucleases such as XRN-1 or Exonuclease T; deadenylases such as HNT3; protein domains responsible for nonsense mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP1, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridin
  • endonucleases e.
  • effector partner comprises a chromatin-modifying enzyme.
  • the effector partner chemically modifies a target nucleic acid, for example by methylating, demethylating, or acetylating the target nucleic acid in a sequence specific or non-specific manner.
  • Base Editing Enzymes [0157]
  • effector partners edit a nucleobase of a target nucleic acid.
  • the effector partner is referred to as a base editing enzyme.
  • a base editing enzyme variant that differs from a naturally occurring base editing enzyme, but it is understood that any reference to a base editing enzyme herein also refers to a base editing enzyme variant.
  • a base editor is a system comprising an effector protein and a base editing enzyme.
  • the base editor comprises a base editing enzyme and an effector protein as independent components.
  • the base editor comprises a fusion protein comprising a base editing enzyme fused or linked to an effector protein.
  • the amino terminus of the effector partner is linked to the carboxy terminus of the effector protein by the linker.
  • the carboxy terminus of the effector partner is linked to the amino terminus of the effector protein by the linker.
  • the base editor is functional when the effector protein is coupled to a guide nucleic acid.
  • the base editor is functional when the effector protein is coupled to a target nucleic acid.
  • the guide nucleic acid imparts sequence specific activity to the base editor.
  • the effector protein comprises a catalytically inactive effector protein (e.g., a catalytically inactive variant of an effector protein described herein).
  • the base editing enzyme comprises deaminase activity. Additional base editors are described herein. [0159] In some embodiments, base editing enzymes catalyze editing (e.g., a chemical modification) of a nucleobase of a nucleic acid molecule, such as DNA or RNA (single stranded or double stranded).
  • a base editing enzyme and therefore a base editor, is capable of converting an existing nucleobase to a different nucleobase, such as: an adenine (A) to guanine (G); cytosine (C) to thymine (T); cytosine (C) to guanine (G); uracil (U) to cytosine (C); guanine (G) to adenine (A); hydrolytic deamination of an adenine or adenosine, or methylation of cytosine (e.g., CpG, CpA, CpT or CpC).
  • base editing In the context of base editing, a person skilled in the art would recognize that reference to the nucleobase (e.g., adenine) or nucleotide (e.g., adenosine) that is being modified by the base editor or base editing enzyme is the nucleobase of the molecule. Accordingly, in the context of base editing, reference to a nucleobase and nucleotide are used interchangeably.
  • base editing enzymes edit a nucleobase on a ssDNA. In some embodiments, base editing enzymes edit a nucleobase on both strands of dsDNA. In some embodiments, base editing enzymes edit a nucleobase of an RNA.
  • a base editing enzyme itself binds or does not bind to the nucleic acid molecule containing the nucleobase.
  • a base pairing between the guide nucleic acid and target strand leads to displacement of a small segment of ssDNA in an “R-loop”.
  • DNA bases within the R-loop are edited by the base editing enzyme having the deaminase enzyme activity.
  • base editing systems for improved efficiency in eukaryotic cells comprise a base editing enzyme, and a catalytically inactive effector protein that generates a nick in the non-edited strand and induce repair of the non-edited strand using the edited strand as a template.
  • a base editing enzyme comprises a deaminase enzyme.
  • Exemplary deaminases are described in US20210198330, WO2021041945, WO2021050571A1, and WO2020123887, all of which are incorporated herein by reference in their entirety.
  • Exemplary deaminase domains are described WO 2018027078 and WO2017070632, and each are hereby incorporated in its entirety by reference.
  • deaminase domains are described in Komor et al., Nature, 533, 420-424 (2016); Gaudelli et al., Nature, 551, 464-471 (2017); Komor et al., Science Advances, 3:eaao4774 (2017), and Rees et al., Nat Rev Genet. 2018 Dec;19(12):770-788. Doi: 10.1038/s41576-018-0059-l, which are hereby incorporated by reference in their entirety.
  • the deaminase functions as a monomer.
  • the deaminase functions as heterodimer with an additional protein.
  • base editing enzymes comprise a DNA glycosylase inhibitor (e.g., an uracil glycosylase inhibitor (UGI) or uracil N-glycosylase (UNG)).
  • the effector partner is a deaminase, e.g., ADAR1/2, ADAR-2, AID, or any functional variant thereof.
  • the base editor is a cytosine base editor (CBE), wherein the base editing enzyme is a cytosine base editing enzyme.
  • the cytosine base editing enzyme, and therefore CBE converts a cytosine to a thymine.
  • a cytosine base editing enzyme accepts ssDNA as a substrate but does not cleave dsDNA, wherein the CBE comprises a catalytically inactive effector protein.
  • a cytosine base editing enzyme introduces a premature stop codon into a target nucleic acid. Accordingly, in some embodiments, a cytosine base editing enzyme is useful in gene knockout application.
  • the catalytically inactive effector protein of the CBE when bound to its cognate DNA, performs local denaturation of the DNA duplex to generate an R-loop in which the DNA strand not paired with a guide nucleic acid exists as a disordered single-stranded bubble.
  • the catalytically inactive effector protein generated ssDNA R-loop enables the CBE to perform efficient and localized cytosine deamination in vitro.
  • deamination activity is exhibited in a window of about 4 to about 10 base pairs.
  • the catalytically inactive effector protein presents a target site to the cytosine base editing enzyme in high effective molarity, which enables the CBE to deaminate cytosines located in a variety of different sequence motifs, with differing efficacies.
  • the CBE mediates RNA-programmed deamination of target cytosines in vitro or in vivo.
  • the cytosine base editing enzyme is a cytidine deaminase. In some embodiments, the cytosine base editing enzyme is a cytosine base editing enzyme described by Koblan et al. (2016) Nature Biotechnology 36:843-846; Komor et al. (2016) Nature 533:420-424; Koblan et al. (2021) “Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning,” Nature Biotechnology; Kurt et al. (2021) Nature Biotechnology 39:41-46; Zhao et al. (2021) Nature Biotechnology 39:35-40; and Chen et al.
  • the effector partner comprises a uracil glycosylase inhibitor (UGI).
  • the CBE described herein comprises UGI.
  • Base excision repair (BER) of U•G in DNA is initiated by a uracil N-glycosylase (UNG), which recognizes a U•G mismatch generated by a CBE and cleaves the glycosidic bond between a uracil and a deoxyribose backbone of DNA.
  • BER results in the reversion of the U•G intermediate created by the cytosine base editing enzyme back to a C•G base pair.
  • the UNG is inhibited by fusion of a UGI to the effector protein.
  • the UGI is a small protein from bacteriophage PBS.
  • the UGI is a DNA mimic that potently inhibits both human and bacterial UNG.
  • the UGI inhibitor is any protein or polypeptide that inhibits UNG.
  • the CBE described herein mediates efficient base editing in bacterial cells and moderately efficient editing in mammalian cells, enabling conversion of a C•G base pair to a T•A base pair through a U•G intermediate.
  • the CBE is modified to increase base editing efficiency while editing more than one strand of DNA.
  • the CBE described herein nicks a non-edited DNA strand.
  • the non-edited DNA strand nicked by the CBE biases cellular repair of a U•G mismatch to favor a U•A outcome, elevating base editing efficiency.
  • a base editor described herein comprising one or more base editing enzymes (e.g., APOBEC1,nickase, and UGI) that efficiently edits in mammalian cells, while minimizing frequency of non-target indels.
  • base editors do not comprise a functional fragment of the base editing enzyme.
  • base editors do not comprise a function fragment of a UGI, where such a fragment excises a uracil residue from DNA by cleaving an N-glycosidic bond.
  • the effector partner comprises a non-protein uracil-DNA glycosylase inhibitor (npUGI).
  • npUGI is selected from a group of small molecule inhibitors of uracil-DNA glycosylase (UDG), or a nucleic acid inhibitor of UDG.
  • the npUGI is a small molecule derived from uracil.
  • the base editor is a cytosine base editor, wherein the based editing enzyme is a cytosine base editing enzyme.
  • the cytosine base editing enzyme is a cytidine deaminase.
  • the base editor comprising the cytidine deaminase is generated by ancestral sequence reconstruction as described in WO2019226953, which is hereby incorporated by reference in its entirety.
  • Non-limiting exemplary cytidine deaminases suitable for use with effector proteins described herein include: APOBEC1, APOBEC2, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, APOBEC3A, BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, and saBE4-Gam as described in WO2021163587, WO2021087246, WO2021062227, and WO2020123887, which are incorporated herein by reference in their entirety.
  • a base editor is a cytosine to guanine base editor (CGBE), wherein the base editing enzyme is a cytosine to guanine base editing enzyme. In some embodiments, the CGBE, converts a cytosine into a guanine.
  • a base editor is an adenine base editor (ABE), wherein the base editing enzyme is an adenine base editing enzyme. In some embodiments, the adenine base editing enzyme, and therefore the ABE, converts an adenine to a guanine. In some embodiments, the adenine base editing enzyme converts an A•T base pair to a G•C base pair.
  • the adenine base editing enzyme converts a target A•T base pair to G•C in vivo or in vitro.
  • the adenine base editing enzymes provided herein reverse spontaneous cytosine deamination, which has been linked to pathogenic point mutations.
  • the adenine base editing enzymes provided herein enable correction of pathogenic SNPs ( ⁇ 47% of disease-associated point mutations).
  • the adenine comprises exocyclic amine that has been deaminated (e.g., resulting in altering its base pairing preferences). In some embodiments, deamination of adenosine yields inosine.
  • inosine exhibits the base-pairing preference of guanine in the context of a polymerase active site, although inosine in the third position of a tRNA anticodon pairs with A, U, or C in mRNA during translation.
  • Non-limiting exemplary adenine base editing enzymes suitable for use with effector proteins described herein include: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), and BtAPOBEC2.
  • Non-limiting exemplary ABEs suitable for use herein include: ABE7, ABE8.1m, ABE8.2m, ABE8.3m, ABE8.4m, ABE8.5m, ABE8.6m, ABE8.7m, ABE8.8m, ABE8.9m, ABE8.10m, ABE8.11m, ABE8.12m, ABE8.13m, ABE8.14m, ABE8.15m, ABE8.16m, ABE8.17m, ABE8.18m, ABE8.19m, ABE8.20m, ABE8.21m, ABE8.22m, ABE8.23m, ABE8.24m, ABE8.1d, ABE8.2d, ABE8.3d, ABE8.4d, ABE8.5d, ABE8.6d, ABE8.7d, ABE8.8d, ABE8.9d, ABE8.10d, ABE8.11d, ABE8.12d, ABE8.13
  • the adenine base editing enzyme is an adenine base editing enzyme described in Chu et al., (2021) The CRISPR Journal 4:2:169-177, incorporated herein by reference.
  • the adenine deaminase is an adenine deaminase described by Koblan et al. (2016) Nature Biotechnology 36:848-846, incorporated herein by reference.
  • the adenine base editing enzyme is an adenine base editing enzyme described by Tran et al. (2020) Nature Communications 11:4871. [0171]
  • the ABE described herein targets polyA signals, splice site acceptors, and start codons.
  • an adenine base editing enzyme is an adenosine deaminase.
  • Non- limiting exemplary adenosine base editors suitable for use herein include ABE9.
  • the ABE comprises an engineered adenosine deaminase enzyme acts on ssDNA.
  • the engineered adenosine deaminase enzyme comprises an adenosine deaminase variant that differs from a naturally occurring deaminase.
  • the adenosine deaminase variant comprises one or more amino acid alterations, including a V82S alteration, a T166R alteration, a Y147T alteration, a Y147R alteration, a Q154S alteration, a Y123H alteration, a Q154R alteration, or a combination thereof.
  • the base editor comprises an adenine deaminase (e.g., TadA).
  • the adenosine deaminase is a TadA monomer (e.g., Tad*7.10, TadA*8 or TadA*9).
  • the adenosine deaminase is a TadA*8 variant (e.g., any one of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24 as described in WO2021163587 and WO2021050571, which are each hereby incorporated by reference in its entirety).
  • the base editor comprises TadA.
  • a base editing enzyme is a deaminase dimer.
  • the ABE comprises the effector protein, the adenine base editing enzyme and the deaminase dimer.
  • the deaminase dimer comprises an adenosine deaminase.
  • the deaminase dimer comprises TadA and a suitable adenine base editing enzyme including an: ABE8e, ABE8.20m, APOBEC3A, Anc APOBEC (a.k.a. AncBE4Max), BtAPOBEC2, and variants thereof.
  • adenine base editing enzyme is fused to amino-terminus or the carboxy- terminus of TadA.
  • a base editor is an RNA base editor, wherein the base editing enzyme is an RNA base editing enzyme.
  • the RNA base editing enzyme comprises an adenosine deaminase.
  • ADAR proteins bind to RNAs and alter their sequence by changing an adenosine into an inosine.
  • RNA base editors comprise an effector protein that is activated by or binds RNA.
  • base editing enzymes, and therefore base editors are used for treating a subject having or a subject suspected of having a disease related to a gene of interest.
  • base editing enzymes, and therefore base editors are useful for treating a disease or a disorder caused by a point mutation in a gene of interest.
  • compositions, systems, and methods described herein comprise a base editor and a guide nucleic acid, wherein the base editor comprises an effector protein and a base editing enzyme, and wherein the guide nucleic acid directs the base editor to a sequence in a target gene.
  • an effector partner provides enzymatic activity that modifies a protein associated with a target nucleic acid.
  • the protein comprises a histone, an RNA binding protein, or a DNA binding protein.
  • methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), Vietnamese histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1); demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID
  • HMT histone methyltransfer
  • effector partners include, but are not limited to, a protein that directly and/or indirectly provides for increased or decreased transcription and/or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.).
  • effector partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.
  • effector partners activate or increase expression of a target nucleic acid.
  • effector partners increase expression of the target nucleic acid relative to its expression in the absence of the effector partners. Relative expression, including transcription and RNA levels, in some embodiments, is assessed, quantified, and compared, e.g., by RT-qPCR.
  • effector partners comprise a transcriptional activator.
  • the transcriptional activators promote transcription by: recruitment of other transcription factor proteins; modification of target DNA such as demethylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones; or a combination thereof.
  • Non-limiting examples of effector partners that promote or increase transcription include: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, D
  • effector partners include: proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for stimulation of RNA splicing (e.g., Serine/Arginine-rich (SR) domains); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat).
  • effector partners inhibit or reduce expression of a target nucleic acid.
  • effector partners reduce expression of the target nucleic acid relative to its expression in the absence of the effector partners.
  • Relative expression including transcription and RNA levels, In some embodiments, is assessed, quantified, and compared, e.g., by RT-qPCR.
  • effector partners comprise a transcriptional repressor.
  • the transcriptional repressors inhibit transcription by: recruitment of other transcription factor proteins; modification of target DNA such as methylation; recruitment of a DNA modifier; modulation of histones associated with target DNA; recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones; or a combination thereof.
  • Non-limiting examples of effector partners that decrease or inhibit transcription include: transcriptional repressors such as the Krüppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SI
  • effector partners include: proteins and protein domains responsible for repressing translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for repression of RNA splicing (e.g., PTB, Sam68, and hnRNP A1); proteins and protein domains responsible for reducing the efficiency of transcription (e.g., FUS (TLS)).
  • fusion proteins comprising the described effector partners and an effector protein are referred to as CRISPRa fusions, wherein the effector partners activate or increase expression of a target nucleic acid.
  • fusion proteins comprising the described effector partners and an effector protein are referred to as CRISPRi fusions, wherein the effector partners inhibit or reduce expression of a target nucleic acid.
  • fusion proteins are targeted by a guide nucleic acid (e.g., guide RNA) to a specific location in a target nucleic acid and exert locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or changes a local chromatin status (e.g., when a fusion sequence is used that edits the target nucleic acid or modifies a protein associated with the target nucleic acid).
  • a guide nucleic acid e.g., guide RNA
  • locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or changes a local chromatin status (e.g., when a fusion sequence is used that edits the target nucleic acid or modifies
  • the modifications are transient (e.g., transcription repression or activation). In some embodiments, the modifications are inheritable. For example, epigenetic modifications made to a target nucleic acid, or to proteins associated with the target nucleic acid, e.g., nucleosomal histones, in a cell, can be observed in a successive generation.
  • effector partner comprises an RNA splicing factor. In some embodiments, the RNA splicing factor is used (in whole or as fragments thereof) for modular organization, with separate sequence-specific RNA binding modules and splicing effector domains.
  • the RNA splicing factors comprise members of the Serine/ Arginine-rich (SR) protein family containing N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion.
  • RRMs N-terminal RNA recognition motifs
  • ESEs exonic splicing enhancers
  • ESSs exonic splicing silencers
  • the RNA splicing factors regulate alternative use of splice site (ss) by binding to regulatory sequences between two alternative sites.
  • ASF/SF2 recognizes ESEs and promote the use of intron proximal sites.
  • hnRNP Al binds to ESSs and shift splicing towards the use of intron distal sites.
  • One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes.
  • Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions.
  • Long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long- lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals.
  • Short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes).
  • a ratio of the two Bcl-x splicing isoforms is regulated by multiple c ⁇ -elements that are located in either core exon region or exon extension region (i.e., between the two alternative 5' splice sites).
  • core exon region or exon extension region i.e., between the two alternative 5' splice sites.
  • effector partners comprise a recombinase.
  • a recombinase system comprising effector proteins described herein and the recombinase.
  • the effector proteins have reduced nuclease activity or no nuclease activity.
  • the recombinase is a site-specific recombinase.
  • the recombinase system comprises a catalytically inactive effector protein, wherein the recombinase can be a site-specific recombinase. Such systems can be used for site- directed transgene insertion.
  • Non-limiting examples of site-specific recombinases include a tyrosine recombinase (e.g., Cre, Flp or lambda integrase), a serine recombinase (e.g., gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase and integrase), or mutants or variants thereof.
  • the recombinase is a serine recombinase.
  • Non-limiting examples of serine recombinases include gamma-delta resolvase, Tn3 resolvase, Sin resolvase, Gin invertase, Hin invertase, Tn5044 resolvase, IS607 transposase, and IS607 integrase.
  • the site-specific recombinase is an integrase.
  • integrases include:Bxb1, wBeta, BL3, phiR4, A118, TG1, MR11, phi370, SPBc, TP901-1, phiRV, FC1, K38, phiBT1, and phiC31.
  • the fusion protein comprises a linker that links the recombinase to the Cas-CRISPR domain of the effector protein.
  • the linker is The-Ser.
  • Linkers for peptides [0187]
  • a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide. Accordingly, in some embodiments, effector proteins, effector partners, or combinations thereof are connected by linkers.
  • the linker comprises or consists of a covalent bond.
  • the linker comprises or consists of a chemical group. In some embodiments, the linker comprises an amino acid. In some embodiments, a peptide linker comprises at least two amino acids linked by an amide bond. In general, the linker connects a terminus of the effector protein to a terminus of the effector partner. In some embodiments, carboxy terminus of the effector protein is linked to the amino terminus of the fusion effector. In some embodiments, carboxy terminus of the effector partner is linked to the amino terminus of the effector protein. In some embodiments, the effector protein and the effector partner are directly linked by a covalent bond. [0188] In some embodiments, linkers comprise one or more amino acids.
  • linker is a protein. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through an amide bond. In some embodiments, a terminus of the effector protein is linked to a terminus of the effector partner through a peptide bond. In some embodiments, linkers comprise an amino acid. In some embodiments, linkers comprise a peptide. In some embodiments, an effector protein is coupled to an effector partner by a linker protein. In some embodiments, the linker comprises any of a variety of amino acid sequences. In some embodiments, the linker comprises a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof.
  • linker comprises a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof.
  • the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility.
  • design of a peptide conjugated to any desired element comprises linkers that are all or partially flexible, such that the linker comprises a flexible linker as well as one or more portions that confer less flexible structure.
  • Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length.
  • linked amino acids described herein comprise at least two amino acids linked by an amide bond.
  • linkers are produced by using synthetic, linker-encoding oligonucleotides to couple proteins, or are encoded by a nucleic acid sequence encoding a fusion protein (e.g., an effector protein coupled to an effector partner).
  • the linker is from 1 to 300, from 1 to 250, from 1 to 200, from 1 to 150, from 1 to 100, from 1 to 50, from 1 to 25, from 1 to 10, from 10 to 300, from 10 to 250, from 10 to 200, from 10 to 150, from 10 to 100, from 10 to 50, from 10 to 25, from 25 to 300, from 25 to 250, from 25 to 200, from 25 to 150, from 25 to 100, from 25 to 50, from 50 to 300, from 50 to 250, from 50 to 200, from 50 to 150, from 50 to 100, from 100 to 300, from 100 to 250, from 100 to 200, from 100 to 150, from 150 to 300, from 150 to 250, from 150 to 200, from 200 to 300, from 200 to 250, or from 250 to 300 amino acids in length.
  • the linker is from 1 to 100 amino acids in length. In some embodiments, the linker is more 100 amino acids in length. In some embodiments, the linker is from 10 to 27 amino acids in length.
  • linker proteins include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n (SEQ ID NO: 801), GGSGGS n (SEQ ID NO: 802), and GGGS n (SEQ ID NO: 803), where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers.
  • linkers comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 804), GGSGG (SEQ ID NO: 805), GSGSG (SEQ ID NO: 806), GSGGG (SEQ ID NO: 807), GGGSG (SEQ ID NO: 808), and GSSSG (SEQ ID NO: 809).
  • the linker comprises one or more repeats a tri-peptide GGS.
  • the linker is a GS-rich linker.
  • the GS-rich linker comprises a peptide having two amino acids (2aa), three amino acids (3aa), five amino acids (5aa), ten amino acids (10aa), twenty amino acids (20aa), or forty amino acids (40aa).
  • the linker is an XTEN linker.
  • the XTEN linker is an XTEN80 linker.
  • the XTEN linker is an XTEN40 linker.
  • the XTEN linker is an XTEN20 linker.
  • the XTEN20 linker has an amino acid sequence of GSGGSPAGSPTSTEEGTSESATPGSG (SEQ ID NO: 781).
  • the XTEN linker is an XTEN10 linker.
  • a polypeptide described herein comprises an activity (e.g., a binding activity, a catalytic activity, or a combination thereof) for a target nucleic acid comprising a target strand and a non-target strand.
  • a length of the linker effects preference of the polypeptide for the activity on the target strand relative to the activity on the non-target strand.
  • a length of the linker effects preference of the polypeptide for the activity on the target strand relative to the activity on the non-target strand, wherein the polypeptide comprises C-terminus of an effector protein described herein linked by the linker to an effector protein described herein.
  • a shorter length of the linker favors activity of the polypeptide on the target strand relative to activity on the non-target strand, wherein the polypeptide comprises C-terminus of an effector protein described herein linked by the linker to an effector protein described herein.
  • a length of a linker effects activity of the polypeptide described herein.
  • a length of the linker effects activity of the polypeptide, wherein the polypeptide comprises N-terminus of an effector protein described herein linked by the linker to an effector protein described herein.
  • linkers do not comprise an amino acid.
  • linkers do not comprise a peptide.
  • linkers comprise a nucleotide, a polynucleotide, a polymer, or a lipid.
  • a linker comprises a polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacrylamide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • POE polyoxyethylene
  • polyurethane polyphosphazene
  • polysaccharides dextran
  • polyvinyl alcohol polyvinylpyrrolidones
  • polyvinyl ethyl ether polyacrylamide
  • polyacrylate polycyanoacrylates
  • lipid polymers chitins, hyaluronic acid,
  • a guide nucleic acid comprises an aptamer.
  • the aptamer serves a similar function as a linker, bringing an effector protein and an effector partner protein into proximity.
  • the aptamer functionally connects two proteins (e.g., effector protein, effector partner, fusion protein, or combinations thereof) by interacting non-covalently with both, thereby bringing both proteins into proximity of the guide nucleic acid.
  • the first protein and/or the second protein comprise or is covalently linked to an aptamer binding moiety.
  • the aptamer is a short single stranded DNA (ssDNA) or RNA (ssRNA) molecule is bound by the aptamer binding moiety.
  • the aptamer is a molecule that mimicks antibody binding activity.
  • the aptamer is classified as a chemical antibody.
  • the aptamer described herein refers to artificial oligonucleotides that bind one or more specific molecules.
  • aptamers exhibit a range of affinities (K D in the pM to ⁇ M range) with little or no off-target binding.
  • proteins (e.g., effector protein, effector partner, fusion protein, or combinations thereof) described herein have been modified (also referred to as an engineered protein).
  • a modification of the proteins comprises addition of one or more amino acids, deletion of one or more amino acids, substitution of one or more amino acids, or combinations thereof.
  • the proteins disclosed herein are engineered proteins. Unless otherwise indicated, reference to the proteins throughout the present disclosure include engineered proteins thereof.
  • proteins (e.g., effector protein, effector partner, fusion protein, or combinations thereof) described herein can be modified with the addition of one or more heterologous peptides.
  • a heterologous peptide comprises at least one subcellular localization signal.
  • a subcellular localization signal can be a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • the NLS facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.
  • the subcellular localization signal is a nuclear export signal (NES), a sequence to keep the protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast or an ER retention signal.
  • the protein described herein is not modified with a subcellular localization signal so that the protein is not targeted to the nucleus, which can be advantageous depending on the circumstance (e.g., when the target nucleic acid is an RNA that is present in the cytosol).
  • a heterologous peptide comprises a chloroplast transit peptide (CTP), also referred to as a chloroplast localization signal or a plastid transit peptide, which targets the protein to a chloroplast.
  • CTP chloroplast transit peptide
  • Chromosomal transgenes from bacterial sources require a sequence encoding a CTP sequence fused to a sequence encoding an expressed protein (e.g., effector protein, effector partner, fusion protein, or combinations thereof) if the expressed protein is to be compartmentalized in the plant plastid (e.g., chloroplast).
  • the CTP is removed in a processing step during translocation into the plastid.
  • the heterologous peptide is an endosomal escape peptide (EEP).
  • EEP is an agent that quickly disrupts the endosome in order to minimize the amount of time that a delivered molecule, such protein, spends in the endosome-like environment, and to avoid getting trapped in the endosomal vesicles and degraded in the lysosomal compartment.
  • An exemplary EEP is set forth in TABLE 2.
  • the heterologous peptide is a cell penetrating peptide (CPP), also known as a Protein Transduction Domain (PTD).
  • CPP cell penetrating peptide
  • PTD Protein Transduction Domain
  • a CPP or PTD is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • heterologous peptides include, but are not limited to, proteins (or fragments/domains thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pil1/Aby1, etc.).
  • a heterologous peptide comprises a protein tag.
  • the protein tag is referred to as purification tag or a fluorescent protein.
  • the protein tag is detectable for use in detection of the protein and/or purification of the protein.
  • compositions, systems and methods comprise a protein tag or use thereof.
  • Any suitable protein tag may be used depending on the purpose of its use.
  • protein tags include a fluorescent protein, a histidine tag, e.g., a 6XHis tag (SEQ ID NO: 810); a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and maltose binding protein (MBP).
  • the protein tag is a portion of MBP that can be detected and/or purified.
  • Non-limiting examples of fluorescent proteins include green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, and tdTomato.
  • a heterologous peptide is located at or near the amino terminus (N- terminus) of the protein (e.g., effector protein, effector partner, fusion protein, or combinations thereof) disclosed herein. In some embodiments, a heterologous peptide is located at or near the carboxy terminus (C-terminus) of the proteins disclosed herein. In some embodiments, a heterologous peptide is located internally in the protein described herein (i.e., is not at the N- or C- terminus of the protein described herein) at a suitable insertion site.
  • protein e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • protein e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the N-terminus, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more heterologous peptides at or near the C-terminus, or a combination of these (e.g., one or more heterologous peptides at the amino- terminus and one or more heterologous peptides at the carboxy terminus).
  • heterologous peptide When more than one heterologous peptide is present, each is selected independently of the others, such that a single heterologous peptide is present in more than one copy and/or in combination with one or more other heterologous peptides present in one or more copies.
  • a heterologous peptide is considered near the N- or C-terminus when the nearest amino acid of the heterologous peptide is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • a heterologous peptide described herein comprises a heterologous peptide sequence recited in TABLE 2.
  • effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the amino acid sequences recited in TABLE 1 and further comprises one or more of the amino acid sequences set forth in TABLE 2.
  • a heterologous peptide described herein comprises an effector partner as described en supra.
  • effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the amino acid sequences recited in TABLE 1 and further comprises an amino acid sequence of SEQ ID NO: 267 at N-terminus of the effector protein.
  • effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the amino acid sequences recited in TABLE 1 and further comprises an amino acid sequence of SEQ ID NO: 4 at C-terminus of the effector protein.
  • effector proteins described herein comprise an amino acid sequence that is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of the amino acid sequences recited in TABLE 1 and further comprises an amino acid sequence of SEQ ID NO: 267 at N-terminus and an amino acid sequence of SEQ ID NO: 4 at C-terminus of the effector protein.
  • proteins (e.g., effector protein, effector partner, fusion protein, or combinations thereof) described herein are encoded by a codon optimized nucleic acid.
  • a nucleic acid sequence encoding the protein described herein is codon optimized.
  • the proteins described herein is codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell.
  • the effector protein is codon optimized for a human cell.
  • proteins e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • proteins comprise one or more modifications that provide altered activity as compared to an activity of naturally-occurring counterpart (e.g., a naturally-occurring nuclease or nickase which is a naturally-occurring protein).
  • activity e.g., nickase, nuclease, binding, deaminase activity
  • proteins described herein is measured relative to a naturally-occurring protein or compositions containing the same in a cleavage assay.
  • proteins e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • proteins comprise one or more modifications that provide increased activity (e.g., catalytic or binding activity) as compared to a naturally-occurring counterpart.
  • proteins provide increased catalytic activity (e.g., nickase, nuclease, deaminase activity) as compared to a naturally- occurring counterpart.
  • proteins provide enhanced nucleic acid binding activity (e.g., enhanced binding of a guide nucleic acid, and/or target nucleic acid) as compared to a naturally- occurring counterpart.
  • proteins have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, or more increase of the activity of a naturally-occurring counterpart.
  • proteins e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • proteins comprise one or more modifications that provide reduced activity (e.g., catalytic or binding activity) as compared to a naturally-occurring counterpart.
  • proteins have a 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less decrease of the activity of a naturally occurring counterpart.
  • decreased activity comprises decreased catalytic activity (e.g., nickase, nuclease, deaminase activity) as compared to a naturally- occurring counterpart.
  • dCAS Proteins an effector protein that has decreased catalytic activity is referred to as catalytically or enzymatically inactive, catalytically or enzymatically dead, as a dead protein or a dCas protein.
  • such a protein comprises an enzymatically inactive domain (e.g., inactive nuclease domain).
  • a nuclease domain e.g., RuvC domain, HNH domain
  • a catalytically inactive effector protein binds to a guide nucleic acid and/or a target nucleic acid but does not cleave the target nucleic acid.
  • a catalytically inactive effector protein associates with a guide nucleic acid to activate or repress transcription of a target nucleic acid.
  • compositions, systems, and methods comprise a fusion protein or uses thereof.
  • a fusion protein generally comprises at least one effector protein, at least one effector partner, or a combination thereof.
  • the effector partner is fused or linked to the effector protein.
  • the effector partner is fused to the N-terminus of the effector protein.
  • the effector partner is fused to the C-terminus of the effector protein.
  • the fusion proteins are multimeric proteins.
  • the multimeric protein is a homomeric protein. In some embodiments, the multimeric protein is a heteromeric protein. In some embodiments, the fusion protein comprising the effector partner is an effector protein. Accordingly, in such embodiments, the fusion protein can comprise at least two effector proteins that are same. In some embodiments, the fusion protein comprises at least two effector proteins that are different. Unless otherwise indicated, reference to effector proteins throughout the present disclosure include fusion proteins described herein. [0212] In some embodiments, the effector partner is a heterologous protein imparting some function or activity that is not provided by an effector protein. In some embodiments, the effector partner is cleaves or modifies the target nucleic acid.
  • the fusion protein disclosed herein provide cleavage activity, such as cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, other activity, or a combination thereof.
  • fusion proteins disclosed herein comprise a RuvC domain comprising cleavage activity.
  • fusion proteins disclosed herein cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA).
  • fusion proteins cleave the target nucleic acid at the target sequence or adjacent to the target sequence.
  • the fusion protein complexes with a guide nucleic acid and the complex interacts with the target nucleic acid.
  • the interaction comprises one or more of: recognition of a protospacer adjacent motif (PAM) sequence within the target nucleic acid by the effector protein, hybridization of the guide nucleic acid to the target nucleic acid, modification of the target nucleic acid by the fusion protein, or combinations thereof.
  • recognition of a PAM sequence within a target nucleic acid directs the modification activity of a fusion protein.
  • modification activity of a fusion protein described herein comprises cleavage activity, binding activity, insertion activity, substitution activity, and the like.
  • modification activity of an effector protein results in: cleavage of at least one strand of a target nucleic acid, deletion of one or more nucleotides of a target nucleic acid, insertion of one or more nucleotides into a target nucleic acid, substitution of one or more nucleotides of a target nucleic acid with an alternative nucleotide, more than one of the foregoing, or any combination thereof.
  • an ability of a fusion protein to edit a target nucleic acid depends upon the effector protein being complexed with a guide nucleic acid, the guide nucleic acid being hybridized to a target sequence of the target nucleic acid, the distance between the target sequence and a PAM sequence, or combinations thereof.
  • a target nucleic acid comprises a target strand and a non-target strand.
  • the fusion protein edits a target strand and/or a non-target strand of a target nucleic acid.
  • the fusion protein described herein comprises a heterologous amino acid sequence that affects formation of a multimeric complex of the fusion protein.
  • the fusion protein comprises an effector protein described herein and an effector partner comprising a Calcineurin A tag, wherein the fusion protein dimerizes in the presence of Tacrolimus (FK506).
  • the fusion protein comprises an effector protein described herein and a SpyTag configured to dimerize or associate with another effector protein in a multimeric complex. Multimeric complex formation is further described herein. IV. Multimeric Complexes [0216] Compositions, systems, and methods of the present disclosure comprise a multimeric complex or uses thereof, wherein the multimeric complex comprises one or more effector proteins that non- covalently interact with one another.
  • a multimeric complex comprises enhanced activity relative to the activity of any one of its effector proteins alone.
  • a multimeric complex comprises two effector proteins (e.g., in dimeric form), wherein the multimeric complex comprises greater nucleic acid binding affinity and/or nuclease activity than that of either of the effector proteins provided in monomeric form.
  • a multimeric complex comprises one or more heterologous proteins fused to one or more effector proteins, wherein the fusion proteins comprise different activity than that of the one or more effector proteins.
  • a multimeric complex comprises an effector protein and a partner protein, wherein the multimeric complex comprises an effector partner, and wherein the multimeric complex comprises greater nucleic acid binding affinity and/or nuclease activity than that of either of the effector protein or effector partner provided in monomeric form.
  • a multimeric complex comprises an affinity for a target sequence of a target nucleic acid and comprises catalytic activity (e.g., cleaving, nicking, inserting or otherwise editing the nucleic acid) at or near the target sequence.
  • a multimeric complex comprises an affinity for a donor nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking, editing or otherwise modifying the nucleic acid by creating cuts) at or near one or more ends of the donor nucleic acid.
  • multimeric complexes are active when complexed with a guide nucleic acid.
  • multimeric complexes are active when complexed with a target nucleic acid.
  • multimeric complexes are active when complexed with a guide nucleic acid, a target nucleic acid, and/or a donor nucleic acid.
  • the multimeric complex cleaves the target nucleic acid.
  • the multimeric complex nicks the target nucleic acid.
  • Various aspects of the present disclosure include compositions and methods comprising multiple polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof), and uses thereof, respectively.
  • An effector protein comprising an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of the amino acid sequences of TABLE 1 are provided with a second effector protein.
  • Two effector proteins target different nucleic acid sequences.
  • multimeric complexes comprise at least one polypeptide (e.g., effector protein, effector partner, fusion protein, or combinations thereof) as described herein.
  • the multimeric complex is a dimer comprising a first polypeptide and a second polypeptide.
  • the first polypeptide and the second polypeptide comprise identical amino acid sequences. In some embodiments, the first polypeptide and the second polypeptide comprise amino acid sequences that are at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to each other. In some embodiments, the first polypeptide and the second polypeptide comprise similar amino acid sequences. In some embodiments, the first polypeptide and the second polypeptide comprise amino acid sequences that are at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% similar to each other.
  • the multimeric complex is a heterodimeric complex comprising at least two polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof) of different amino acid sequences.
  • the multimeric complex comprises two, three, four, five, six, seven, eight, nine, or ten polypeptides.
  • the multimeric complex is a heterodimeric complex comprising a first effector protein and a second effector protein, wherein the amino acid sequence of the first effector protein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% identical to the amino acid sequence of the second effector protein.
  • At least one effector protein of the multimeric complex comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of the amino acid sequences of TABLE 1.
  • each effector protein of the multimeric complex independently comprises an amino acid sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to any one of the amino acid sequences of TABLE 1.
  • the multimeric complex described herein targets polyA signals, splice site acceptors, and start codons.
  • the multimeric complex cannot create stop codons for knock-down.
  • the multimeric complex is a dimer comprising fusion protein described herein.
  • the fusion protein comprises the effector protein described herein, and the effector partner described herein.
  • the dimer is formed due to non-covalent interactions between the effector proteins of monomers.
  • N- and C- termini of “formerly active” monomer is closer to 5’ region of non-target strand, while the termini of the “other” monomer is closer to 3’ region, which results in a larger editing window of the multimeric complex having a larger editing window on the non-target strand.
  • the multimeric complex has a lower editing window for a target strand due to inaccessibility for the effector partner.
  • Protospacer Adjacent Motif (PAM) Sequences [0222]
  • a polypeptide (e.g., effector protein, effector partner, fusion protein or combinations thereof) of the present disclosure cleaves or nicks a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand.
  • cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides of a 5’ or 3’ terminus of a PAM sequence.
  • polypeptides described herein recognize a PAM sequence.
  • recognizing a PAM sequence comprises interacting with a sequence adjacent to the PAM.
  • a target nucleic acid comprises a target sequence that is adjacent to a PAM sequence.
  • the polypeptide does not require a PAM to bind and/or cleave a target nucleic acid.
  • a target nucleic acid is a single stranded target nucleic acid comprising a target sequence.
  • the single stranded target nucleic acid comprises a PAM sequence described herein that is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) or directly adjacent to the target sequence.
  • an RNP cleaves the single stranded target nucleic acid.
  • a target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, wherein the target strand comprises a target sequence.
  • the PAM sequence is located on the target strand. In some embodiments, the PAM sequence is located on the non-target strand. In some embodiments, the PAM sequence described herein is adjacent (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides) to the target sequence on the target strand or the non-target strand. In some embodiments, the PAM sequence is located 5’ of a reverse complement of the target sequence on the non-target strand. In some embodiments, such a PAM described herein is directly adjacent to the target sequence on the target strand or the non-target strand. In some embodiments, an RNP cleaves the target strand or the non- target strand.
  • the RNP cleaves both, the target strand and the non-target strand. In some embodiments, an RNP recognizes the PAM sequence, and hybridizes to a target sequence of the target nucleic acid. In some embodiments, the RNP cleaves the target nucleic acid, wherein the RNP has recognized the PAM sequence and is hybridized to the target sequence of the target nucleic acid and, optionally, modifies the target nucleic acid.
  • an effector protein described herein, or a multimeric complex thereof recognizes a PAM on a target nucleic acid. In some embodiments, multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid.
  • an effector protein of the present disclosure, or a multimeric complex thereof cleaves or nicks a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a 5’ or 3’ terminus of a PAM sequence.
  • PAM protospacer adjacent motif
  • compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid. Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid.
  • Guide nucleic acids are also referred to herein as “guide RNA.”
  • a guide nucleic acid, as well as any components thereof comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more engineered modifications as described herein), or any combinations thereof.
  • nucleotide sequences described herein may be described as a nucleotide sequence of either DNA or RNA, however, no matter the form the nucleotide sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the nucleotide sequence that encodes a guide nucleic acid, such as a nucleotide sequence described herein for a vector.
  • nucleotide sequences described herein also discloses the complementary nucleotide sequence, the reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which can be a nucleotide sequence for use in a guide nucleic acid as described herein.
  • a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the nucleotide sequences described herein.
  • Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • a nucleotide “U” is a uracil or a 1N-Methyl-Pseudouridine.
  • a guide nucleic acid comprises a naturally occurring sequence.
  • a guide nucleic acid comprises a non-naturally occurring sequence, wherein the nucleotide sequence of the guide nucleic acid, or any portion thereof, is different from the nucleotide sequence of a naturally occurring guide nucleic acid.
  • a guide nucleic acid of the present disclosure comprises one or more of the following: a) a single nucleic acid molecule; b) a DNA base; c) an RNA base; d) a modified base; e) a modified sugar; f) a modified backbone; and the like. Modifications are described herein and throughout the present disclosure (e.g., in the section entitled “Engineered Modifications”).
  • a guide nucleic acid is chemically synthesized or recombinantly produced by any suitable methods.
  • guide nucleic acids and portions thereof are found in or identified from a CRISPR array present in the genome of a host organism or cell.
  • a portion of the guide nucleic acid (e.g., spacer sequence) has a degree of complementarity to the target sequence and hybridizes to the target sequence.
  • the guide nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence.
  • the guide nucleic acid comprises at least 10 contiguous nucleotides that are complementary to the target sequence in the target nucleic acid.
  • guide nucleic acid comprises a spacer sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target sequence.
  • a guide nucleic acid comprises a first region that is not complementary to a target sequence of a target nucleic acid (FR) and a second region is complementary to the target sequence of the target nucleic acid (SR), wherein the FR and the SR are heterologous to each other.
  • FR is located 5’ to SR (FR-SR).
  • SR is located 5’ to FR (SR-FR).
  • the FR comprises one or more repeat sequence, handle sequence, intermediary sequence, or combinations thereof. In some embodiments, at least a portion of the FR interacts or binds to an effector protein.
  • the SR comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g., has complementarity with, or can hybridize to a target sequence in) a target nucleic acid.
  • the first region, the second region, or both are about 8 linked nucleotides, about 10 linked nucleotides, about 12 linked nucleotides, about 14 linked nucleotides, about 16 linked nucleotides, about 18 linked nucleotides, about 20 linked nucleotides, about 22 linked nucleotides, about 24 linked nucleotides, about 26 linked nucleotides, about 28 linked nucleotides, about 30 linked nucleotides, about 32 linked nucleotides, about 34 linked nucleotides, about 36 linked nucleotides, about 38 linked nucleotides, about 40 linked nucleotides, about 42 linked nucleotides, about 44 linked nucleotides, about 46 linked nucleotides, about 48 linked nucleotides, or about 50 linked nucleotides.
  • the first region, the second region, or both comprise from about 8 to about 12, from about 8 to about 16, from about 8 to about 20, from about 8 to about 24, from about 8 to about 28, from about 8 to about 30, from about 8 to about 32, from about 8 to about 34, from about 8 to about 36, from about 8 to about 38, from about 8 to about 40, from about 8 to about 42, from about 8 to about 44, from about 8 to about 48, or from about 8 to about 50 linked nucleotides.
  • the first region, the second region, or both comprise a GC content of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%.
  • the first region, the second region, or both comprise a GC content of from about 1% to about 95%, from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, from about 20% to about 60%, from about 25% to about 50%, or from about 30% to about 40%.
  • the first region, the second region, or both have a melting temperature of about 38 °C, about 40 °C, about 42 °C, about 44 °C, about 46 °C, about 48 °C, about 50 °C, about 52 °C, about 54 °C, about 56 °C, about 58 °C, about 60 °C, about 62 °C, about 64 °C, about 66 °C, about 68 °C, about 70 °C, about 72 °C, about 74 °C, about 76 °C, about 78 °C, about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, about 90 °C, or about 92 °C.
  • the first region, the second region, or both have a melting temperature of from about 35 °C to about 40 °C, from about 35 °C to about 45 °C, from about 35 °C to about 50 °C, from about 35 °C to about 55 °C, from about 35 °C to about 60 °C, from about 35 °C to about 65 °C, from about 35 °C to about 70 °C, from about 35 °C to about 75 °C, from about 35 °C to about 80 °C, or from about 35 °C to about 85 °C.
  • compositions, systems, and methods of the present disclosure further comprise an additional nucleic acid, wherein a portion of the additional nucleic acid at least partially hybridizes to the first region of the guide nucleic acid.
  • the additional nucleic acid is at least partially hybridized to the 5’ end of the second region of the guide nucleic acid.
  • an unhybridized portion of the additional nucleic acid at least partially, interacts with an effector protein or polypeptide.
  • the compositions, systems, and methods of the present disclosure comprise a dual nucleic acid system comprising the guide nucleic acid and the additional nucleic acid as described herein.
  • the guide nucleic acid also forms complexes as described through herein.
  • a guide nucleic acid hybridizes to another nucleic acid, such as target nucleic acid, or a portion thereof.
  • a guide nucleic acid complexes with an effector protein.
  • a guide nucleic acid-effector protein complex is described herein as an RNP.
  • at least a portion of the complex binds, recognizes, and/or hybridizes to a target nucleic acid.
  • a guide nucleic acid and an effector protein are complexed to form an RNP
  • at least a portion of the guide nucleic acid hybridizes to a target sequence in a target nucleic acid.
  • a RNP hybridizes to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein (e.g., PAM) or to modify and/or recognize non-target sequences depending on the guide nucleic acid, and in some embodiments, the effector protein, used.
  • a guide nucleic acid comprises or forms intramolecular secondary structure (e.g., hairpins, stem-loops, etc.).
  • a guide nucleic acid comprises a stem- loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • the guide nucleic acid comprises a pseudoknot (e.g., a secondary structure comprising a stem, at least partially, hybridized to a second stem or half-stem secondary structure).
  • an effector protein recognizes a guide nucleic acid comprising multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the guide nucleic acid comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • the compositions, systems, and methods of the present disclosure comprise two or more guide nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 9, 10 or more guide nucleic acids), and/or uses thereof.
  • multiple guide nucleic acids target an effector protein to different locations in the target nucleic acid by hybridizing to different target sequences.
  • a first guide nucleic acid hybridizes within a location of the target nucleic acid that is different from where a second guide nucleic acid hybridizes the target nucleic acid.
  • the first loci and the second loci of the target nucleic acid are located at least 1, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides apart.
  • the first loci and the second loci of the target nucleic acid are located between 100 and 200, 200 and 300, 300 and 400, 400 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900 or 900 and 1000 nucleotides apart. In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an intron of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid are located in an exon of a gene. In some embodiments, the first loci and/or the second loci of the target nucleic acid span an exon-intron junction of a gene.
  • compositions, systems, and methods comprise a donor nucleic acid that is inserted in replacement of a deleted or cleaved sequence of the target nucleic acid.
  • compositions, systems, and methods comprising multiple guide nucleic acids or uses thereof comprise multiple effector proteins, wherein the effector proteins are identical, non-identical, or combinations thereof.
  • a guide nucleic acid comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides.
  • a guide nucleic acid comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides.
  • the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleotides.
  • a guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence.
  • a eukaryotic sequence is a nucleotide sequence that is present in a host eukaryotic cell.
  • Such a nucleotide sequence is distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses.
  • Said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, a non-coding (e.g., promoter or enhancer) region, a selectable marker, tag, signal, and the like.
  • a target sequence is a eukaryotic sequence.
  • a length of a guide nucleic acid is about 30 to about 120 linked nucleotides.
  • the length of a guide nucleic acid is about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a guide nucleic acid is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. In some embodiments, the length of a guide nucleic acid is greater than about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides.
  • the length of a guide nucleic acid is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, or about 125 linked nucleotides.
  • guide nucleic acids comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid.
  • the elements comprise one or more nucleotide alterations, nucleotide sequences, intermolecular secondary structures, or intramolecular secondary structures (e.g., one or more hair pin regions, one or more bulges, etc.).
  • guide nucleic acids comprise one or more linkers connecting different nucleotide sequences as described herein.
  • a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides.
  • a linker comprises any suitable linker, examples of which are described herein.
  • guide nucleic acids comprise one or more nucleotide sequences as described herein (e.g., TABLE 4, TABLE 4.1, TABLE 5, TABLE 5.1, TABLE 6, TABLE 7, TABLE 8, TABLE 14, TABLE 15 and TABLE 16).
  • nucleotide sequences described herein are described as a nucleotide sequence of either DNA or RNA, however, no matter the form of the nucleotide sequence described, it is readily understood that such nucleotide sequences may be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the nucleotide sequence that encodes a guide nucleic acid, such as a nucleotide sequence described herein for a vector.
  • nucleotide sequences described herein also discloses the complementary nucleotide sequence, the reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which is a nucleotide sequence for use in a guide nucleic acid as described herein.
  • guide nucleic acids described herein comprise at least two nucleotide sequences as described herein (e.g., TABLE 9).
  • guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the nucleotide sequences described herein.
  • Alternative nucleotides may be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • a guide nucleic acid comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of the sequences of TABLE 14, TABLE 15 and TABLE 16.
  • a guide nucleic acid comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides of any one of the sequences of TABLE 14, TABLE 15 and TABLE 16.
  • the guide nucleic acid comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, wherein the target nucleic acid is any one of: a naturally occurring eukaryotic sequence, a naturally occurring prokaryotic sequence, a naturally occurring viral sequence, a naturally occurring bacterial sequence, a naturally occurring fungal sequence, an engineered eukaryotic sequence, an engineered prokaryotic sequence, an engineered viral sequence, an engineered bacterial sequence, an engineered fungal sequence, a fragment of a naturally occurring sequence, a fragment of an engineered sequence, and combinations thereof.
  • the guide nucleic acid is isolated from any one of: a naturally occurring cell, a eukaryotic cell, a prokaryotic cell, a plant cell, a fungal cell, an animal cell, cell of an invertebrate, a fly cell, a cell of a vertebrate, a mammalian cell, a primate cell, a non-human primate cell, a human cell, a living cell, a non-living cell, a modified cell, a derived cell, and a non-naturally occurring cell.
  • Spacer Sequence [0249]
  • guide nucleic acids described herein comprise one or more spacer sequences.
  • a spacer sequence hybridizes to a target sequence of a target nucleic acid.
  • a spacer sequence comprises a nucleotide sequence that is, at least partially, hybridizable to an equal length of a sequence (e.g., a target sequence) of a target nucleic acid. Exemplary hybridization conditions are described herein.
  • the spacer sequence functions to direct an RNP complex comprising the guide nucleic acid to the target nucleic acid for detection and/or modification.
  • the spacer sequence functions to direct a RNP to the target nucleic acid for detection and/or modification.
  • a spacer sequence is complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein described herein.
  • a spacer sequence comprises at least 5 to about 50 contiguous nucleotides that are complementary to a target sequence in a target nucleic acid.
  • a spacer sequence comprises at least 5 to about 50 linked nucleotides.
  • a spacer sequence comprises at least 5 to about 50, at least 5 to about 25, at least about 10 to about 25, or at least 15 to about 25 linked nucleotides.
  • the spacer sequence comprises 15-28 linked nucleotides.
  • a spacer sequence comprises 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides.
  • the spacer sequence comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides.
  • a spacer sequence is adjacent to a repeat sequence.
  • a spacer sequence follows a repeat sequence in a 5’ to 3’ direction.
  • a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction.
  • the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequences. In some embodiments, linkers comprise any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions.
  • a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid.
  • a spacer sequence hybridizes to an equal length portion of a target nucleic acid (e.g., a target sequence).
  • a target nucleic acid such as DNA or RNA, comprises a cancer gene or gene associated with a genetic disorder, or an amplicon thereof, as described herein.
  • a target nucleic acid is a gene selected from TABLE 11.
  • a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid selected from TABLE 11.
  • a target nucleic acid is a nucleic acid associated with a disease or syndrome set forth in TABLE 13.
  • a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of a target nucleic acid associated with a disease or syndrome set forth in TABLE 13.
  • the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that hybridize to the target sequence. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to the target sequence. [0253] It is understood that the spacer sequence of a spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence.
  • the spacer sequence in some embodiments, comprises at least one alteration, such as a substituted or modified nucleotide, that is not complementary to the corresponding nucleotide of the target sequence.
  • Spacer sequences are further described throughout herein.
  • a spacer sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the spacer sequences of TABLE 4, TABLE 4.1, TABLE 5 and TABLE 5.1.
  • the spacer sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20, or at least 21 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 4, TABLE 4.1, TABLE 5 and TABLE 5.1.
  • the spacer sequence comprises one or more nucleotide alterations at one or more positions in the nucleotide sequence recited in TABLE 4, TABLE 4.1, TABLE 5 and TABLE 5.1.
  • Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • guide nucleic acids described herein comprise one or more repeat sequences.
  • a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid.
  • a repeat sequence comprises a nucleotide sequence that interacts with an effector protein.
  • a repeat sequence is connected to another sequence of a guide nucleic acid, such as an intermediary sequence, that non-covalently interacts with an effector protein.
  • a repeat sequence includes a nucleotide sequence that forms a guide nucleic acid-effector protein complex (e.g., a RNP complex).
  • the repeat sequence is between 10 and 50, 12 and 48, 14 and 46, 16 and 44, and 18 and 42 nucleotides in length. In some embodiments, the repeat sequence is at least 13, at least 15, at least 18, at least 21, at least 24, at least 27, at least 30, at least 33, or at least 36 linked nucleotides in length. [0257] In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is preceded by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence.
  • a repeat sequence is 3’ to an intermediary sequence.
  • an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction.
  • a repeat sequence is linked to a spacer sequence and/or an intermediary sequence.
  • a guide nucleic acid comprises a repeat sequence linked to a spacer sequence and/or to an intermediary sequence by a direct link or by any suitable linker, examples of which are described herein. [0258]
  • guide nucleic acids comprise more than one repeat sequence (e.g., two or more, three or more, or four or more repeat sequences).
  • a guide nucleic acid comprises more than one repeat sequence separated by another sequence of the guide nucleic acid.
  • a guide nucleic acid comprises two repeat sequences, wherein the first repeat sequence is followed by a spacer sequence, and the spacer sequence is followed by a second repeat sequence in the 5’ to 3’ direction.
  • the more than one repeat sequences are identical.
  • the more than one repeat sequences are not identical.
  • the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex).
  • the two sequences are not directly linked and hybridize to form a stem loop structure.
  • the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp). In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence comprises a bulge.
  • the repeat sequence comprises a hairpin or stem- loop structure, optionally at the 5’ portion of the repeat sequence.
  • a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary. In some embodiments, such sequences comprise 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity).
  • a guide nucleic acid comprises a nucleotide sequence that, when involved in hybridization events, hybridizes over one or more segments of a target nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.).
  • a repeat sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to an equal length portion of any one of the repeat sequences in TABLE 6.
  • the repeat sequence is at least 85% identical to any one of sequences set forth in TABLE 6.
  • a repeat sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 6.
  • a repeat sequence comprises one or more nucleotide alterations at one or more positions in the nucleotide sequence recited in TABLE 6.
  • Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers.
  • the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers.
  • the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers.
  • the guide nucleic acid comprises more than one linker. In some embodiments, at least two of the more than one linker are the same.
  • a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides.
  • the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides.
  • a linker comprises a nucleotide sequence recited in TABLE 7.
  • a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences. In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker. Intermediary sequence [0265] In some embodiments, guide nucleic acids described herein comprise one or more intermediary sequences. In some embodiments, intermediary RNA is a nucleotide sequence in a handle sequence, wherein the nucleotide sequence, at least partially, is non-covalently bound to an effector protein forming a complex (e.g., an RNP complex).
  • a complex e.g., an RNP complex
  • an intermediary sequence used in the present disclosure is not transactivated or transactivating.
  • an intermediary sequence is also be referred to as an intermediary RNA, although it comprises deoxyribonucleotides instead of or in addition to ribonucleotides, and/or modified bases.
  • the intermediary sequence non- covalently binds to an effector protein.
  • the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.
  • a length of the intermediary sequence is at least 30, 40, or 50 linked nucleotides.
  • a length of the intermediary sequence is not greater than 30, 40, 50 or 60 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 60, about 40 to about 60, or about 50 to about 60 linked nucleotides.
  • an intermediary sequence also comprises or forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). In some embodiments, an intermediary sequence comprises from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region.
  • the 5’ region hybridizes to the 3’ region. In some embodiments, the 5’ region of the intermediary sequence does not hybridize to the 3’ region.
  • the hairpin region comprises a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.
  • an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure).
  • an effector protein interacts with an intermediary sequence comprising a single stem region or multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • an intermediary sequence comprises 1, 2, 3, 4, 5 or more stem regions.
  • an intermediary sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to the intermediary sequence in TABLE 8.
  • an intermediary sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or at least 50 contiguous nucleotides of the intermediary sequence recited in TABLE 8.
  • an intermediary sequence comprises one or more nucleotide alterations at one or more positions in the nucleotide sequence recited in TABLE 8.
  • guide nucleic acids described herein comprise one or more handle sequences.
  • the handle sequence comprises an intermediary sequence. In such instances, at least a portion of an intermediary sequence non-covalently bonds with an effector protein.
  • the intermediary sequence is at the 3’-end of the handle sequence. In some embodiments, the intermediary sequence is at the 5’- end of the handle sequence.
  • the handle sequence further comprises one or more of linkers and repeat sequences.
  • an intermediary sequence and repeat sequence are directly linked (e.g., covalently linked, such as through a phosphodiester bond).
  • the intermediary sequence and repeat sequence are linked by a suitable linker, examples of which are provided herein.
  • the linker comprises the nucleotide sequence recited in TABLE 7.
  • the intermediary sequence is 5’ to the repeat sequence.
  • the intermediary sequence is 5’ to the linker.
  • the intermediary sequence is 3’ to the repeat sequence.
  • a single guide nucleic acid also referred to as a single guide RNA (sgRNA)
  • sgRNA single guide RNA
  • a handle sequence comprises or forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region).
  • handle sequences comprise a stem-loop structure comprising a stem region and a loop region.
  • the stem region is 4 to 8 linked nucleotides in length.
  • the stem region is 5 to 6 linked nucleotides in length.
  • the stem region is 4 to 5 linked nucleotides in length.
  • the handle sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure).
  • an effector protein recognizes a handle sequence comprising multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another.
  • the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others.
  • the handle sequence comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • a length of the handle sequence is at least 30, 40, 50, 60, or 70 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 40, 50, 60, or 70 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 70, about 40 to about 70, about 50 to about 70, or about 60 to about 70 linked nucleotides.
  • a handle sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the handle sequences in TABLE 9.
  • a handle sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 contiguous nucleotides of any one of the handle sequences recited in TABLE 9.
  • a handle sequence comprises one or more nucleotide alterations at one or more positions in the nucleotide sequence recited in TABLE 9.
  • Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • compositions, systems and methods described herein comprise a single nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins.
  • a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein.
  • a second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid.
  • the effector protein is not transactivated by the guide nucleic acid. In other words, activity of effector protein does not require binding to a second non-target nucleic acid molecule.
  • An exemplary guide nucleic acid for a single nucleic acid system is a crRNA or a sgRNA.
  • a guide nucleic acid comprises a crRNA.
  • the guide nucleic acid is the crRNA.
  • a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence.
  • the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)).
  • the repeat sequence and the spacer sequence are connected by a linker.
  • a crRNA is useful as a single nucleic acid system for compositions, methods, and systems described herein or as part of a single nucleic acid system for compositions, methods, and systems described herein.
  • a crRNA is useful as part of a single nucleic acid system for compositions, methods, and systems described herein.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA, wherein a repeat sequence of the crRNA interacts with an effector protein.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA linked to another nucleotide sequence that is non-covalently bound by an effector protein.
  • a repeat sequence of a crRNA can be linked to an intermediary sequence.
  • a single nucleic acid system comprises a guide nucleic acid comprising a crRNA and an intermediary sequence.
  • a crRNA comprises deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a crRNA comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides.
  • a crRNA comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides.
  • the length of the crRNA is about 20 to about 120 linked nucleotides.
  • the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides.
  • a crRNA sequence comprises a repeat sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences set forth in TABLE 6, and a spacer sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences recited in TABLE 4, TABLE 4.1, TABLE 5 and TABLE 5.1.
  • a crRNA sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the repeat sequences recited in TABLE 6, and at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the spacer sequences recited in TABLE 4, TABLE 4.1, TABLE 5 and TABLE 5.1.
  • a guide nucleic acid comprises a sgRNA.
  • a guide nucleic acid is a sgRNA.
  • a sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence.
  • the handle sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)).
  • the handle sequence and the spacer sequence are connected by a linker.
  • a sgRNA comprises one or more of a handle sequence, an intermediary sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof.
  • a sgRNA comprises a handle sequence and a spacer sequence; an intermediary sequence and an crRNA; an intermediary sequence, a repeat sequence and a spacer sequence; and the like.
  • a sgRNA comprises an intermediary sequence and an crRNA.
  • an intermediary sequence is 5’ to a crRNA in a sgRNA.
  • a sgRNA comprises a linked intermediary sequence and crRNA.
  • an intermediary sequence and a crRNA are linked in a sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a crRNA are linked in a sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises a handle sequence and a spacer sequence.
  • a handle sequence is 5’ to a spacer sequence in a sgRNA.
  • a sgRNA comprises a linked handle sequence and spacer sequence.
  • a handle sequence and a spacer sequence are linked in a sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • a handle sequence and a spacer sequence are linked in a sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence.
  • an intermediary sequence is 5’ to a repeat sequence in a sgRNA.
  • a sgRNA comprises a linked intermediary sequence and repeat sequence.
  • an intermediary sequence and a repeat sequence are linked in a sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a repeat sequence are linked in a sgRNA by any suitable linker, examples of which are provided herein.
  • a repeat sequence is 5’ to a spacer sequence in a sgRNA.
  • a sgRNA comprises a linked repeat sequence and spacer sequence.
  • a repeat sequence and a spacer sequence are linked in a sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond)
  • a repeat sequence and a spacer sequence are linked in a sgRNA by any suitable linker, examples of which are provided herein.
  • a sgRNA sequence comprises a nucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences in TABLE 4, TABLE 4.1, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • a sgRNA sequence comprises a handle sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences in TABLE 9, and a spacer sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences recited in TABLE 4, TABLE 4.1 and TABLE 5.
  • a sgRNA sequence comprises a handle sequence comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 9, and a spacer sequence comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 4, TABLE 4.1 and TABLE 5.
  • Polypeptides e.g., effector proteins
  • nucleic acids e.g., engineered guide nucleic acids
  • polypeptides examples include modifications that do not alter the primary sequence of the polypeptides or nucleic acids, such as chemical derivatization of polypeptides (e.g., acylation, acetylation, carboxylation, amidation, etc.), or modifications that do alter the primary sequence of the polypeptide or nucleic acid.
  • polypeptides that have a modified glycosylation pattern e.g., those made by: modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes).
  • polypeptides that have phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, or phosphothreonine).
  • Modifications disclosed herein can also include modification of described polypeptides and/or guide nucleic acids through any suitable method, such as molecular biological techniques and/or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable for their intended purpose (e.g., in vivo administration, in vitro methods, or ex vivo applications).
  • Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring synthetic amino acids. In some embodiments, D-amino acids is substituted for some or all of the amino acid residues. Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. [0289] Modifications can further include the introduction of various groups to polypeptides and/or guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g., an effector protein), which allow for linking to other molecules or to a surface.
  • a polypeptide e.g., an effector protein
  • cysteines are used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
  • Modifications can further include changing of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability.
  • nucleic acids described herein e.g., engineered guide nucleic acids
  • modifications of a nucleic acid include a base editing, a base modification, a backbone modification, a sugar modification, or combinations thereof.
  • the modifications can be of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.
  • nucleic acids e.g., nucleic acids encoding effector proteins, engineered guide nucleic acids, or nucleic acids encoding engineered guide nucleic acids
  • nucleic acids described herein comprise one or more modifications comprising: 2’O-methyl modified nucleotides (e.g., 2’-O-Methyl (2’OMe) sugar modifications); 2’ fluoro modified nucleotides (e.g., 2’-fluoro (2’-F) sugar modifications); locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7-methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phospho
  • repeat sequences described herein comprise at least three nucleotides that are modified. In some embodiments, repeat sequences described herein comprise at least three consecutive nucleotides from the 5’-end of the guide nucleic acid that are modified. In some embodiments, spacer sequences described herein comprise at least three nucleotides that are modified. In some embodiments, spacer sequences described herein comprise at least three consecutive nucleotides from the 3’-end of the guide nucleic acid that are modified. In some embodiments, guide nucleic acids described herein comprise at least three nucleotides that are modified. In some embodiments, guide nucleic acids described herein comprise at least six nucleotides that are modified.
  • guide nucleic acids described herein comprise one or more 2’-O-Methyl (2’OMe) sugar modifications, one or more phosphorothioate (PS) backbone modifications, or combinations thereof.
  • the one or more 2’OMe sugar modification, PS backbone modification, or combinations thereof are contained within a portion of a guide nucleic acid (e.g., repeat sequence) that at least partially interacts with an effector protein described herein.
  • the guide nucleic acids comprise PS backbone modification between -3 and -2 positions of a repeat sequence present in the guide nucleic acid, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • the guide nucleic acids comprise PS backbone modification between -3 and -2 positions of a repeat sequence present in the guide nucleic acid in combination with at least one modification between -16 and -12 positions of the repeat sequence present in the guide nucleic acid, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • the guide nucleic acids comprise a 2’OMe sugar modification at -14 position of a repeat sequence present in the guide nucleic acid, and a PS backbone modification between -3 and -2 positions of the repeat sequence present in the guide nucleic acid, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • the guide nucleic acids comprise a 2’OMe sugar modification at -16 position, and a PS backbone modification between -3 and -2 positions of a repeat sequence present in the guide nucleic acid, wherein the repeat sequence comprises at least 24 nucleotides.
  • the guide nucleic acids comprise PS backbone modifications between -3 and -2 positions of a repeat sequence present in the guide nucleic acid, and - 13 and -12 positions of the repeat sequence, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • the guide nucleic acids comprise PS backbone modifications between -3 and -2 positions of a repeat sequence present in the guide nucleic acid, and -14 and -13 positions of the repeat sequence, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • the guide nucleic acids comprise PS backbone modifications between -3 and -2 positions of a repeat sequence present in the guide nucleic acid, and -15 and -14 positions of the repeat sequence, wherein the repeat sequence comprises a nucleotide length described herein, such as at least 24 nucleotides.
  • guide nucleic acids described herein comprise at least six nucleotides comprising 2’OMe sugar modification and at least five PS backbone modifications.
  • compositions, systems, and methods described herein comprise a vector or a use thereof.
  • a vector can comprise a nucleic acid of interest.
  • the nucleic acid of interest comprises one or more components of a composition or system described herein.
  • the nucleic acid of interest comprises a nucleotide sequence that encodes one or more components of the composition or system described herein.
  • one or more components comprises a polypeptide(s) (e.g., effector protein(s), effector partner(s), fusion protein(s), or combinations thereof), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s).
  • the component comprises a nucleic acid encoding a polypeptide (e.g., effector protein(s), effector partner(s), fusion protein(s), or combinations thereof), a donor nucleic acid, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid.
  • a vector is a part of a vector system.
  • the vector system comprises a library of vectors each encoding one or more component of a composition or system described herein.
  • components described herein e.g., an effector protein, a guide nucleic acid, and/or a donor nucleic acid
  • components described herein are each encoded by different vectors of the system.
  • a vector encoding a donor nucleic acid further encodes a target nucleic acid.
  • a vector comprises a nucleotide sequence encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof as described herein.
  • the one or more polypeptides comprise at least two polypeptides. In some embodiments, the at least two polypeptides are the same.
  • the at least two polypeptides are different from each other.
  • the nucleotide sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell.
  • the vector comprises the nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more polypeptides.
  • a vector encodes one or more of any system components, including but not limited to polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof), guide nucleic acids, and target nucleic acids as described herein.
  • a system component encoding sequence is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell.
  • a vector encodes 1, 2, 3, 4 or more of any system components.
  • a vector encodes two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence.
  • a vector encodes the polypeptide and the guide nucleic acid.
  • a vector encodes the polypeptide, a guide nucleic acid, a donor nucleic acid, or combinations thereof.
  • a vector comprises one or more guide nucleic acids, or a nucleotide sequence encoding the one or more guide nucleic acids as described herein.
  • the one or more guide nucleic acids comprise at least two guide nucleic acids.
  • the at least two guide nucleic acids are the same.
  • the at least two guide nucleic acids are different from each other.
  • the guide nucleic acid or the nucleotide sequence encoding the guide nucleic acid is operably linked to a promoter that is operable in a target cell, such as a eukaryotic cell.
  • the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids.
  • the vector comprises a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more guide nucleic acids.
  • a vector comprises one or more donor nucleic acids as described herein.
  • the one or more donor nucleic acids comprise at least two donor nucleic acids.
  • the at least two donor nucleic acids are the same.
  • the at least two donor nucleic acids are different from each other.
  • the vector comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more donor nucleic acids.
  • a vector comprises or encodes one or more regulatory elements. Regulatory elements, in some embodiments, are refer to as transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide.
  • a vector comprises or encodes for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like.
  • a vector comprises or encodes for one or more elements, such as, for example, ribosome binding sites, and RNA splice sites.
  • Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3′ direction) coding or non-coding sequence.
  • a promoter can be linked at its 3′ terminus to a nucleic acid, the expression or transcription of which is desired, and extends upstream (5′ direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level.
  • a promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”.
  • the promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes.
  • promoters including inducible promoters, are used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest.
  • the nucleic acid of interest can be operably linked to a promoter.
  • promotors comprises any suitable type of promoter envisioned for the compositions, systems, and methods described herein.
  • Examples include constitutively active promoters (e.g., CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc.
  • constitutively active promoters e.g., CMV promoter
  • inducible promoters e.g., heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.
  • spatially restricted and/or temporally restricted promoters e.g., a tissue specific promoter, a cell type specific promoter, etc.
  • Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl).
  • SV40 early promoter mouse mammary tumor virus long terminal repeat (LTR) promoter
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • Hl human Hl promoter
  • a polypeptide e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof
  • vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein.
  • the vector comprises a nucleotide sequence of a promoter.
  • the vector comprises two promoters.
  • the vector comprises three promoters.
  • a length of the promoter is less than about 500, less than about 400, less than about 300, or less than about 200 linked nucleotides.
  • a length of the promoter is at least 100, at least 200, at least 300, at least 400, or at least 500 linked nucleotides.
  • Non-limiting examples of promoters include CMV, 7SK, EF1a, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1-10, H1, TEF1, GDS, ADH1, CaMV35S, HSV TK, Ubi, U6, MNDU3, MSCV, MND and CAG.
  • some promoters e.g., U6, enhanced U6, Hl and 7SK prefers the nucleic acid being transcribed having “g” nucleotide at the 5’ end of the coding sequence.
  • vectors provided herein comprise a promotor driving expression or transcription of any one of the guide nucleic acids described herein (e.g., TABLE 4, TABLE 4.1, TABLE 5, TABLE 5.1, TABLE 6, TABLE 7, TABLE 8, TABLE 9 and TABLE 10) further comprises “g” nucleotide at 5’ end of the guide nucleic acid, wherein the promotor is selected from U6, enhanced U6, Hl and 7SK.
  • the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.
  • the inducible promoter only drives expression of its corresponding coding sequence (e.g., polypeptide or guide nucleic acid) when a signal is present, e.g., a hormone, a small molecule, a peptide.
  • a signal e.g., a hormone, a small molecule, a peptide.
  • Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter.
  • the promoter is an activation-inducible promoter, such as a CD69 promoter.
  • the promoter for expressing a polypeptide is a ubiquitous promoter.
  • the ubiquitous promoter comprises MND or CAG promoter sequence.
  • the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell).
  • the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell).
  • the promoter is EF1a.
  • the promoter is ubiquitin.
  • vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner.
  • a vector described herein is a nucleic acid expression vector.
  • a vector described herein is a recombinant expression vector.
  • a vector described herein is a messenger RNA (mRNA).
  • a vector comprising the recombinant nucleic acid as described herein, wherein the vector is a viral vector, an adeno associated viral (AAV) vector, a retroviral vector, or a lentiviral vector.
  • a vector described herein or a recombinant nucleic acid described herein is comprised in a cell.
  • a vector described herein is a delivery vector.
  • the delivery vector is a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof.
  • the delivery vehicle is a non-viral vector.
  • the delivery vector is a plasmid.
  • the plasmid comprises DNA.
  • the plasmid comprises RNA.
  • the plasmid comprises circular double-stranded DNA.
  • the plasmid is linear.
  • the plasmid comprises one or more coding sequences of interest and one or more regulatory elements.
  • the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria.
  • the plasmid is a minicircle plasmid.
  • the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid.
  • the plasmids are engineered through synthetic or other suitable means known in the art.
  • the genetic elements are assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which is then be readily ligated to another genetic sequence.
  • the plasmid is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the plasmid is formulated for delivery via electroporation.
  • vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription.
  • an administration of a non-viral vector comprises contacting a cell, such as a host cell, with the non-viral vector.
  • a physical method or a chemical method is employed for delivering the vector into the cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery.
  • Exemplary chemical methods include delivery of the recombinant polynucleotide by liposomes such as, cationic lipids or neutral lipids; lipofection; dendrimers; lipid nanoparticle (LNP); or cell-penetrating peptides.
  • a vector is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein.
  • a vector is administered in a single vehicle, such as a single expression vector.
  • At least two of the three components, a nucleic acid encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof), one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acid, are provided in the single expression vector.
  • components, such as a guide nucleic acid and a polypeptide (e.g., effector protein, effector partner, fusion protein, or combinations thereof) are encoded by the same vector.
  • a polypeptide e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • a nucleic acid encoding same or a nucleic acid encoding same
  • an engineered guide nucleic acid or a nucleic acid that, when transcribed, produces same
  • a polypeptides e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • a polypeptides e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • an engineered guide nucleic acid or a nucleic acid that, when transcribed, produces same
  • donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.
  • a vector system is administered as part of a method of nucleic acid detection, editing, and/or treatment as described herein, wherein at least two vectors are co- administered.
  • the at least two vectors comprise different components.
  • the at least two vectors comprise the same component having different sequences.
  • a nucleic acid encoding one or more polypeptides e.g., effector proteins, effector partners, fusion proteins, or combinations thereof
  • one or more donor nucleic acids e.g., a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof
  • the nucleic acid encoding the polypeptides e.g., effector proteins, effector partners, fusion proteins, or combinations thereof
  • a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors.
  • compositions and systems provided herein comprise a lipid or a lipid particle.
  • a lipid particle is a lipid nanoparticle (LNP).
  • a lipid or a lipid nanoparticle can encapsulate a nucleic acid (e.g., DNA or RNA) encoding one or more of the components as described herein.
  • a lipid or a lipid nanoparticle can encapsulate an expression vector as described herein.
  • LNPs are a non-viral delivery system for delivery of the composition and/or system components described herein. LNPs are particularly effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkarni et al., (2016) Nucleic Acid Therapeutics, 28(3):146-157).
  • compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce one or more effector proteins, one or more guide nucleic acids, one or more donor nucleic acids, or any combinations thereof to a cell.
  • Non-limiting examples of lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio- responsive polymers.
  • the ionizable lipids exploits chemical-physical properties of the endosomal environment (e.g., pH) offering improved delivery of nucleic acids.
  • the ionizable lipids are neutral at physiological pH.
  • the ionizable lipids are protonated under acidic pH.
  • the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.
  • a LNP comprises an outer shell and an inner core.
  • the outer shell comprises lipids.
  • the lipids comprise modified lipids.
  • the modified lipids comprise pegylated lipids.
  • the lipids comprise one or more of cationic lipids, anionic lipids, ionizable lipids, and non-ionic lipids.
  • the LNP comprises one or more of N1,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide (TT3), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), 1,2-dimyristoyl-sn- glycerol, methoxypolyethylene glycol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG 2000), derivatives, analogs or variants thereof, or combinations thereof.
  • DOPC 1,2-Dioleoyl-
  • the LNP comprises one or more ionizable lipid.
  • ionizable lipids include, but are not limited to: 4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12- octadecadien-1-yl-10,13-nonadecadien-1-yl ester (DLin-MC3-DMA, CAS No. 1224606-06-7); N,N- dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethanamine (DLin-KC2-DMA, CAS No.
  • 9,12- octadecadienoic acid (9Z,12Z)-1,1′,1′′,1′′′-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1-butanediylnitrilodi- 4,1-butanediyl)] ester (OF-C4-Deg-Lin, CAS No.
  • Arcturus Lipid 2,2 (8,8) 4C CH3 (ATX-0114, CAS No. 2230647-28-4) ); di((Z)-non-2-en-1-yl) 8,8'-((2-((2- (dimethylamino)ethyl)thio)acetyl)azanediyl)dioctanoate (ATX-001, CAS No. 1777792-33-2); di((Z)- non-2-en-1-yl) 8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX-002, CAS No.
  • the LNP comprise a combination of two, three, four, five or more of the foregoing ionizable lipids.
  • the LNP has a negative net overall charge prior to complexation with one or more of a guide nucleic acid, a nucleic acid encoding the one or more guide nucleic acid, a nucleic acid encoding a polypeptide (e.g., effector protein, effector partner, fusion protein, or combinations thereof), and/or a donor nucleic acid.
  • the inner core is a hydrophobic core.
  • the one or more of a guide nucleic acid, the nucleic acid encoding the one or more guide nucleic acid, the nucleic acid encoding the polypeptide, and/or the donor nucleic acid forms a complex with one or more of the cationic lipids and the ionizable lipids.
  • the nucleic acid encoding the polypeptide or the nucleic acid encoding the guide nucleic acid is self-replicating.
  • a LNP comprises one or more of cationic lipids, ionizable lipids, and modified versions thereof.
  • the ionizable lipid comprises N1,N3,N5-tris(3- (didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) or a derivative thereof.
  • the LNP comprises one or more of TT3 and pegylated TT3.
  • the publication WO2016187531 is hereby incorporated by reference in its entirety, which describes representative LNP formulations in Table 2 and Table 3, and representative methods of delivering LNP formulations in Example 7.
  • a LNP comprises a lipid composition targeting to a specific organ.
  • the lipid composition comprises lipids having a specific alkyl chain length that controls accumulation of the LNP in the specific organ (e.g., liver or spleen). In some embodiments, the lipid composition comprises a biomimetic lipid that controls accumulation of the LNP in the specific organ (e.g., brain). In some embodiments, the lipid composition comprises lipid derivatives (e.g., cholesterol derivatives) that controls accumulation of the LNP in a specific cell (e.g., liver endothelial cells, Kupffer cells, hepatocytes).
  • a specific cell e.g., liver endothelial cells, Kupffer cells, hepatocytes.
  • the LNP described herein comprises nucleic acids (e.g., DNA or RNA) encoding an effector protein described herein, an effector partner described herein, a fusion protein described herein, a guide nucleic acid described herein, or combinations thereof.
  • the LNP comprises an mRNA that produces an effector protein described herein, an effector partner described herein, or a fusion protein described herein when translated.
  • the LNP comprises chemically modified guide nucleic acids.
  • LNPs described herein comprises one or more ionizable lipids, phospholipids, cholesterols, and PEG lipids.
  • the LNP comprises one or more ionizable lipids at a molar ratio ranging from 20 to 60, from 25 to 60, from 30 to 60, from 35 to 60, from 40 to 60, from 45 to 60, from 50 to 60, from 55 to 60, from 20 to 55, from 25 to 55, from 30 to 55, from 35 to 55, from 40 to 55, from 45 to 55, or from 50 to 55.
  • the LNP comprises one or more cholesterols at a molar ratio ranging from 5 to 20, from 7 to 20, from 5 to 17, or from 7 to 17.
  • the LNP comprises one or more cholesterols at a molar ratio ranging from 30 to 65, from 35 to 65, from 40 to 65, from 45 to 65, from 50 to 65, from 55 to 65, from 60 to 65, from 30 to 60, from 35 to 60, from 40 to 60, from 45 to 60, from 50 to 60, or from 55 to 60.
  • the LNP comprises one or more PEG lipids at a molar ratio ranging from 0.5 to 5, from 1.5 to 5, from 2.5 to 5, from 0.5 to 4, from 1.5 to 4, from 2.5 to 4, from 0.5 to 3, from 1.5 to 3, or from 2.5 to 3.
  • the LNPs are formulated according to any one of the LNP formulations described in TABLE 21.
  • a vector described herein comprises a viral vector.
  • the viral vector comprises a nucleic acid to be delivered into a host cell by a recombinantly produced virus or viral particle.
  • the nucleic acid comprises single-stranded or double stranded, linear or circular, segmented or non-segmented.
  • the nucleic acid comprises DNA, RNA, or a combination thereof.
  • the vector is an adeno-associated viral vector.
  • viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and ⁇ - retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • retroviruses e.g., lentiviruses and ⁇ - retroviruses
  • adenoviruses e.g., lentiviruses and ⁇ - retroviruses
  • AAVs adeno-associated viruses
  • the viral vector is a recombinant viral vector.
  • the vector is a retroviral vector.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector comprises gamma- retroviral vector.
  • a viral vector provided herein is derived from or based on any such virus.
  • the gamma-retroviral vector is derived from a Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or a Murine Stem cell Virus (MSCV) genome.
  • the lentiviral vector is derived from the human immunodeficiency virus (HIV) genome.
  • the viral vector is a chimeric viral vector.
  • the chimeric viral vector comprises viral portions from two or more viruses.
  • the viral vector corresponds to a virus of a specific serotype.
  • a viral vector is an adeno-associated viral vector (AAV vector).
  • AAV vector adeno-associated viral vector
  • a viral particle that delivers a viral vector described herein is an AAV.
  • the AAV comprises any AAV known in the art.
  • the viral vector corresponds to a virus of a specific AAV serotype.
  • the AAV serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, an AAV-rh10 serotype, and any combination, derivative, or variant thereof.
  • the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.
  • scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA.
  • an AAV vector described herein is a chimeric AAV vector.
  • the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes.
  • a chimeric AAV vector is genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.
  • AAV vector described herein comprises two inverted terminal repeats (ITRs).
  • the viral vector provided herein comprises two inverted terminal repeats of AAV.
  • a nucleotide sequence between the ITRs of an AAV vector provided herein comprises a sequence encoding genome editing tools.
  • the genome editing tools comprise a nucleic acid encoding one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof), a nucleic acid encoding the one or more polypeptides comprising a heterologous peptide (e.g., a nuclear localization signal (NLS), polyA tail), one or more guide nucleic acids, a nucleic acid encoding the one or more guide nucleic acids, respective promoter(s), one or more donor nucleic acid, or any combinations thereof.
  • viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein.
  • a coding region of the AAV vector forms an intramolecular double-stranded DNA template thereby generating the AAV vector that is a self-complementary AAV (scAAV) vector.
  • the scAAV vector comprises the nucleotide sequence encoding genome editing tools that has a length of about 2 kb to about 3 kb.
  • the AAV vector provided herein is a self-inactivating AAV vector.
  • the AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector.
  • methods of producing AAV delivery vectors herein comprise packaging a nucleic acid encoding a polypeptide (e.g., effector protein, effector partner, fusion protein, or combinations thereof) and a guide nucleic acid, or a combination thereof, into an AAV vector.
  • a polypeptide e.g., effector protein, effector partner, fusion protein, or combinations thereof
  • methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid encoding: (i) a guide nucleic acid; (ii) a Replication (Rep) gene; and (iii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging the polypeptide encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector.
  • promoters, stuffer sequences, and any combination thereof are packaged in the AAV vector.
  • the AAV vector is package 1, 2, 3, 4, or 5 guide nucleic acids or copies thereof.
  • the AAV vector comprises inverted terminal repeats, e.g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat.
  • the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.
  • a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes are not the same.
  • the Rep gene and ITR from a first AAV serotype is used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes are not the same.
  • a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein is indicated AAV2/9.
  • the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.
  • AAV particles described herein are recombinant AAV (rAAV).
  • rAAV particles are generated by transfecting AAV producing cells with an AAV- containing plasmid carrying the nucleotide sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA.
  • the AAV producing cells are mammalian cells.
  • host cells for rAAV viral particle production are mammalian cells.
  • a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a variant thereof, or a combination thereof.
  • rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell.
  • producing rAAV virus particles in a mammalian cell comprises transfecting vectors that express the rep protein, the capsid protein, and the gene-of-interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends.
  • rAAV is produced in a non-mammalian cell.
  • rAAV is produced in an insect cell.
  • the insect cell for producing rAAV viral particles comprises a Sf9 cell.
  • production of rAAV virus particles in insect cells comprises infecting the insect cells with baculovirus.
  • production of rAAV virus particles in insect cells comprises infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end.
  • rAAV virus particles are produced by the One Bac system.
  • rAAV virus particles can be produced by the Two Bac system.
  • the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome.
  • an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of- interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Ther., 1;13(16):1935-43; and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety. IX.
  • Target Nucleic Acids Disclosed herein are compositions, systems and methods for detecting and/or editing a target nucleic acid.
  • the target nucleic acid is a double stranded nucleic acid.
  • the target nucleic acid is a single stranded nucleic acid.
  • the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting an RNP.
  • the single stranded nucleic acid comprises a RNA, wherein the RNA comprises a mRNA, a rRNA, a tRNA, a non-coding RNA, a long non-coding RNA, a microRNA (miRNA), and a single-stranded RNA (ssRNA).
  • the target nucleic acid is complementary DNA (cDNA) synthesized from a single-stranded RNA template in a reaction catalyzed by a reverse transcriptase.
  • the target nucleic acid comprises an RNA, a DNA, or combination thereof.
  • guide nucleic acids described herein hybridize to a portion of the target nucleic acid.
  • the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
  • a target nucleic acid comprising a target sequence comprises a PAM sequence.
  • the PAM sequence is adjacent to the target sequence.
  • the PAM sequence is 3’ to the target sequence.
  • the PAM sequence is directly 3’ to the target sequence.
  • the PAM sequence is directly 5’ to the target sequence.
  • the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence.
  • any target nucleic acid of interest that is generated using the methods described herein to comprise a PAM sequence and thus be a PAM target nucleic acid.
  • a PAM target nucleic acid refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by a polypeptide system.
  • a target nucleic acid comprises 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 linked nucleotides.
  • the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 linked nucleotides.
  • the target nucleic acid comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 linked nucleotides.
  • the target nucleic acid comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 linked nucleotides.
  • the target sequence in the target nucleic acid comprises at least 10 contiguous nucleotides that are complementary to the guide nucleic acid or engineered guide nucleic acid.
  • compositions, systems, and methods described herein comprise a target nucleic acid that is responsible for a disease, contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g., contain a unique sequence of nucleotides).
  • the target nucleic acid has undergone a modification (e.g., an editing) after contacting with an RNP.
  • the editing is a change in the nucleotide sequence of the target nucleic acid.
  • the change comprises an insertion, deletion, or substitution of one or more nucleotides compared to the target nucleic acid that has not undergone any modification.
  • a target nucleic acid comprises a portion or a specific region of a nucleic acid from a genomic locus, any DNA amplicon of, a reverse transcribed mRNA, or a cDNA from a gene described herein.
  • the target nucleic acid is an amplicon of at least a portion of a gene.
  • a target nucleic acid comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to any one of the nucleotide sequences recited in TABLE 11, a complement thereof, or a reverse complement thereof.
  • the target nucleic acid comprises a target sequence, wherein the target sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, 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, or at least 25 contiguous nucleotides of the nucleotide sequence recited in TABLE 11.
  • the target sequence comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to an equal length portion of the nucleotide sequence recited in TABLE 11.
  • a target nucleic acid comprises a nucleotide sequence that is present in a gene that is expressed by a liver cell.
  • the nucleotide sequence is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to any one of the nucleotide sequences recited in TABLE 11, a complement thereof, or a reverse complement thereof.
  • the target nucleic acid present in a gene that is expressed by a liver cell comprises a target sequence, wherein the target sequence comprises at least 10, at least 11, at least 12, at least 13, at least 14, 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, or at least 25 contiguous nucleotides of the nucleotide sequence recited in TABLE 11.
  • the target sequence present in a gene that is expressed by a liver cell comprises a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% identical to an equal length portion of the nucleotide sequence recited in TABLE 11.
  • Donor Nucleic Acid insertion [0336] In some embodiments, a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or target sequence. [0337] In some embodiments, a donor nucleic acid comprises a transgene.
  • the transgene comprises a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell.
  • the transgene comprises (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced.
  • a donor nucleic acid can comprise a transgene.
  • the cell in which transgenes expression occur can be a target cell, such as a host cell.
  • the target nucleic acid sequence comprises a nucleotide sequence within a human safe harbor locus.
  • the human safe harbor locus is located in human chromosome 4.
  • the targeting the human safe harbor loci as described herein for incorporation of a transgene as described herein results in stable expression of the functional human protein and wherein the incorporation does not result in insertional oncogenesis.
  • human safe harbor locus is within intron 1 of human ALB gene. Accordingly, in some embodiments, the target nucleic acid is the human ALB gene.
  • the human ALB gene is located at chromosome 4q11- q13.
  • SEQ ID NO: 261 provides a nucleotide sequence of the intron 1 of human ALB gene for use with the compositions, systems and methods of the disclosure.
  • the intron 1 of human ALB gene comprises one or more nucleotide alterations at one or more positions relative to SEQ ID NO: 261.
  • Alternative nucleotides can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • the human safe harbor locus is located in human chromosome 1.
  • a human safe harbor locus is within exon 1-2 of human PCSK9 gene.
  • the human PCSK9 gene is located at chromosome 1p32.3.
  • SEQ ID NO: 514 provides a nucleotide sequence of the exon 1-2 of human PCSK9 gene for use with the compositions, systems and methods of the disclosure.
  • the exon 1-2 of human PCSK9 gene comprises one or more nucleotide alterations at one or more positions relative to SEQ ID NO: 514.
  • the donor nucleic acid comprises single-stranded DNA or linear double- stranded DNA.
  • the donor nucleic acid comprises a nucleotide sequence encoding a functional polypeptide and/or wherein the donor nucleic acid comprises a wildtype sequence.
  • the donor nucleic acid comprises a protein coding sequence, a gene, a gene fragment, an exon, an intron, an exon fragment, an intron fragment, a gene regulatory fragment, a gene regulatory region fragment, coding sequences thereof, or combinations thereof.
  • the donor nucleic acid comprises a naturally occurring sequence. In some embodiments, the naturally occurring sequence does not contain a mutation. [0341] In some embodiments, the donor nucleic acid comprises a gene fragment, an exon fragment, an intron fragment, a gene regulatory region fragment, or combinations thereof. In some embodiments, the fragment is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, or at least 80 contiguous nucleotides. [0342] In some embodiments, a donor nucleic acid of any suitable size is integrated into a target nucleic acid or a genome.
  • the donor nucleic acid integrated into the target nucleic acid or the genome is less than 3, about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 kilobases in length.
  • the donor nucleic acid is more than 500 kilobases (kb) in length.
  • a viral vector comprising a donor nucleic acid introduces the donor nucleic acid into a cell following transfection.
  • the donor nucleic acid is introduced into the cell by any mechanism of the transfecting viral vector, including, but not limited to, integration into the genome of the cell or introduction of an episomal plasmid or viral genome.
  • an effector protein as described herein facilitates insertion of a donor nucleic acid at a site of cleavage or between two cleavage sites by cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break – nuclease activity.
  • a donor nucleic acid serves as a template in the process of homologous recombination, which carries an alteration that is to be or has been introduced into a target nucleic acid.
  • the genetic information including the alteration, is copied into the target nucleic acid by way of homologous recombination.
  • a donor nucleic acid is inserted at a cleavage site within the target nucleic acid, wherein the cleavage site is generated by an effector protein or fusion protein described herein.
  • the donor nucleic acid encodes an amino acid sequence of a functional human protein.
  • the functional human protein has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 180%, at least 200%, at least 300%, at least 400% enzymatic activity compared to wildtype.
  • the functional human protein comprises a wildtype protein.
  • Specific examples of functional human proteins include wildtype and engineered versions of proteins that are deficient, under-expressed or aberrantly expressed in certain human subjects. In some embodiments, these subjects suffer from disorders that result in deficiency or aberrant expression of these proteins.
  • a functional human protein comprises any one of the proteins selected from CFTR, DMD, A1AT, GAA, FXN, F8, F9, SOD1, C9, HTT, MECP2, SMN1, TARDBP, FUS, RHO, and USH2A, wherein the protein is understood to be poorly expressed in subjects with genetic disorders, such as monogenic disorders.
  • Exemplary amino acid sequences of functional human proteins are provided in TABLE 12.
  • the wildtype protein comprises human wildtype protein sequence.
  • the human protein comprises an amino acid sequence that is associated with any of disease recited in TABLE 13.
  • target nucleic acids described herein comprise a mutation.
  • a composition, system or method described herein can be used to edit a target nucleic acid comprising a mutation such that the mutation is edited to be the wild-type nucleotide or nucleotide sequence.
  • a composition, system or method described herein can be used to detect a target nucleic acid comprising a mutation.
  • a mutation results in the insertion of at least one amino acid in a protein encoded by the target nucleic acid.
  • a mutation results in the deletion of at least one amino acid in a protein encoded by the target nucleic acid.
  • a mutation results in the substitution of at least one amino acid in a protein encoded by the target nucleic acid.
  • a mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid results in misfolding of a protein encoded by the target nucleic acid.
  • a mutation results in a premature stop codon, thereby resulting in a truncation of the encoded protein.
  • Non-limiting examples of mutations are insertion-deletion (indel), a point mutation, single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation or variation, and frameshift mutations.
  • an indel mutation is an insertion or deletion of one or more nucleotides.
  • a point mutation comprises a substitution, insertion, or deletion.
  • a frameshift mutation occurs when the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region.
  • a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation of one or more nucleotides.
  • a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat.
  • an SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. In some embodiments, an SNP is associated with altered phenotype from wild type phenotype. In some embodiments, the SNP is a synonymous substitution or a nonsynonymous substitution. In some embodiments, the nonsynonymous substitution is a missense substitution or a nonsense point mutation. In some embodiments, the synonymous substitution is a silent substitution. In some embodiments, the mutation is associated with one or more of protein expression, protein activity, and protein structural stability. [0351] In some embodiments, a target nucleic acid described herein comprises a mutation of one or more nucleotides.
  • the one or more nucleotides comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.
  • the mutation comprises a deletion, insertion, and/or substitution of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides.
  • the mutation comprises a deletion, insertion, and/or substitution of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides.
  • the mutation is located in a non-coding region or a coding region of a gene, wherein the gene is a target nucleic acid.
  • a mutation is in an open reading frame of a target nucleic acid.
  • guide nucleic acids described herein hybridize to a portion of the target nucleic acid comprising or adjacent to the mutation.
  • the target nucleic acid comprises one or more mutations.
  • the target nucleic acid comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations as compared to the unmutated target nucleic acid.
  • the target nucleic acid comprises a sequence comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations as compared to the wildtype sequence.
  • the target nucleic acid comprises a mutation associated with a disease or disorder.
  • target nucleic acids comprise a mutation, wherein the mutation is a SNP.
  • the single nucleotide mutation or SNP is associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken.
  • the SNP is associated with altered phenotype from wild type phenotype.
  • a single nucleotide mutation, SNP, or deletion described herein is associated with a disease, such as a genetic disease.
  • the SNP is a synonymous substitution or a nonsynonymous substitution.
  • the nonsynonymous substitution is a missense substitution or a nonsense point mutation.
  • the synonymous substitution is a silent substitution.
  • the mutation is a deletion of one or more nucleotides.
  • the single nucleotide mutation, SNP, or deletion is associated with a disease such as a genetic disorder.
  • the mutation such as a single nucleotide mutation, a SNP, or a deletion, is encoded in the nucleotide sequence of a target nucleic acid from the germline of an organism or is encoded in a target nucleic acid from a diseased cell.
  • the mutation is associated with a disease, such as a genetic disorder.
  • the mutation is encoded in the nucleotide sequence of a target nucleic acid from the germline of an organism or is encoded in a target nucleic acid from a diseased cell.
  • a target nucleic acid described herein comprises a mutation associated with a disease.
  • a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to or suffers from, a disease, disorder, condition, or syndrome.
  • a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome.
  • a mutation associated with a disease is also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease.
  • a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, or pathological state.
  • a mutation associated with a disease comprises the co-occurrence of a mutation and the phenotype of a disease.
  • the mutation occurs in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation.
  • a target nucleic acid described herein comprises a mutation associated with a disease, wherein the target nucleic acid is a PCSK9 gene.
  • one or more mutations or aberrant expression of PCSK9 protein is associated, at least in part, to a hypercholesterolemia.
  • one or more mutations in PCSK9 gene results in autosomal dominant familial hypercholesterolemia.
  • PCSK9 protein deficiency can put individual at risk of developing early heart disease.
  • one or more mutations or aberrant expression of PCSK9 protein is associated with heart disease.
  • compositions comprising one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combinations thereof) described herein or nucleic acids encoding the one or more polypeptides, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or combinations thereof.
  • repeat sequences of the one or more guide nucleic acids interact with the one or more of the effector proteins.
  • spacer sequences of the one or more guide nucleic acids hybridizes with a target sequence of a target nucleic acid.
  • the compositions comprise one or more donor nucleic acids described herein.
  • compositions edit a target nucleic acid in a cell or a subject. In some embodiments, the compositions edit a target nucleic acid or the expression thereof in a cell, in a tissue, in an organ, in vitro, in vivo, or ex vivo. In some embodiments, the compositions edit a target nucleic acid in a sample comprising the target nucleic.
  • compositions described herein comprise plasmids described herein, viral vectors described herein, non-viral vectors described herein, or combinations thereof. In some embodiments, compositions described herein comprise the viral vectors. In some embodiments, compositions described herein comprise an AAV.
  • compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell- penetrating peptides. In some embodiments, compositions described herein comprise an LNP.
  • compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cell- penetrating peptides. In some embodiments, compositions described herein comprise an LNP.
  • compositions described herein comprise an LNP.
  • compositions described herein are formulations of introducing compositions or components of a system described herein to a host.
  • compositions described herein are pharmaceutical compositions. In some embodiments, the pharmaceutical compositions comprise compositions described herein, or systems described herein.
  • the pharmaceutical composition comprises a pharmaceutically acceptable salt, one or more of a vehicle, adjuvant, excipient, or carrier, such as a filler, disintegrant, a surfactant, a binder, a lubricant, or combinations thereof.
  • a vehicle adjuvant, excipient, or carrier
  • a filler such as a filler, disintegrant, a surfactant, a binder, a lubricant, or combinations thereof.
  • Remington The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia; Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988- 1999, Marcel Dekker, New York; and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick, 2015, CRC Press, Boca Raton disclose various carriers used in formulating pharmaceutically acceptably compositions and known techniques for the preparation thereof.
  • Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.
  • buffers e.g., neutral buffered saline, phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose, dextran, mannitol
  • polypeptides or amino acids e.g.
  • the vector is formulated for delivery through injection by a needle carrying syringe. In some embodiments, the composition is formulated for delivery by electroporation. In some embodiments, the composition is formulated for delivery by chemical method. In some embodiments, the pharmaceutical compositions comprise a virus vector or a non-viral vector.
  • Pharmaceutical compositions described herein comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNO 3 . In some embodiments, the salt is Mg 2+ SO 4 2 ⁇ .
  • compositions described herein are in the form of a solution (e.g., a liquid).
  • the solution is formulated for injection, e.g., intravenous or subcutaneous injection.
  • the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.
  • the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5.
  • the pH of the solution is less than 7.
  • the pH is greater than 7.
  • the one or more components individually comprises one or more of the following: (i) an effector protein, or a nucleic acid encoding the effector protein; (ii) an effector partner, or a nucleic acid encoding the effector partner; (iii) a fusion protein, or a nucleic acid encoding the fusion protein; and (iv) a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid.
  • the one or more components comprises at least one of a guide nucleic acid and a nucleic acid encoding the guide nucleic acid.
  • systems are used for modifying or editing a target nucleic acid.
  • systems are used for inserting a donor nucleic acid into a target nucleic acid.
  • systems comprise an effector protein or a nucleic acid encoding the effector protein described herein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid a reagent described herein, a donor nucleic acid described herein, support medium, or a combination thereof.
  • the effector protein comprises an effector protein, or a fusion protein thereof, described herein.
  • effector proteins comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 1.
  • effector proteins comprise an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% similar to any one of the nucleotide sequences recited in TABLE 1.
  • the guide nucleic acid comprises at least one nucleotide sequence selected from the nucleotide sequences recited in any one of TABLE 4, TABLE 4.1, TABLE 5, TABLE 5.1, TABLE 6, TABLE 8 and TABLE 9.
  • the target nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 11.
  • the donor nucleic acid comprises a nucleotide sequence encoding any one of the amino acid sequences recited in TABLE 12.
  • the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that is associated with any one of the diseases recited in TABLE 13.
  • the one or more components individually comprises one or more of the following: (i) an effector protein, or a nucleic acid encoding the effector protein; (ii) an effector partner, or a nucleic acid encoding the effector partner; (iii) a fusion protein, or a nucleic acid encoding the fusion protein; (iv) a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and (v) the donor nucleic acid encoding a transgene that comprises a functional human protein that is expressed upon incorporation into the human safe harbor locus.
  • the safe harbor locus comprises a nucleotide sequence encoding intron 1 gene of human albumin.
  • the target nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 261, or a portion thereof.
  • the safe harbor locus comprises a nucleotide sequence encoding exon 1-2 gene of human PCSK9 gene.
  • the target nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 514, or a portion thereof.
  • the donor nucleic acid comprises a nucleotide sequence encoding any one of the amino acid sequences recited in TABLE 12. In some embodiments, the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence that is associated with any one of the diseases recited in TABLE 13. [0364] In some embodiments, systems comprise an effector protein described herein, a guide nucleic acid described herein, a reagent, support medium, or a combination thereof. In some embodiments, the effector protein comprises an effector protein, or a fusion protein thereof, described herein.
  • effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 1.
  • the guide nucleic acid comprises a spacer sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 4, TABLE 4.1 and TABLE 5.
  • the guide nucleic acid comprises a repeat sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 6.
  • the guide nucleic acid comprises an intermediary sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or 100% identical to the nucleotide sequence recited in TABLE 8.
  • the guide nucleic acid comprises a handle sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or 100% identical to any one of the nucleotide sequences recited in TABLE 9.
  • Amplification Reagents/Components [0365]
  • systems described herein comprise a reagent or component for amplifying a nucleic acid.
  • reagents for amplifying a nucleic acid include polymerases, primers, and nucleotides.
  • systems comprise reagents for nucleic acid amplification of a target nucleic acid in a sample.
  • nucleic acid amplification of the target nucleic acid improves at least one of sensitivity, specificity, or accuracy of the assay in detecting the target nucleic acid.
  • nucleic acid amplification is isothermal nucleic acid amplification, providing for the use of the system or system in remote regions or low resource settings without specialized equipment for amplification.
  • amplification of the target nucleic acid increases the concentration of the target nucleic acid in the sample relative to the concentration of nucleic acids that do not correspond to the target nucleic acid.
  • the reagents for nucleic acid amplification comprise a recombinase, a primer, an oligonucleotide primer, an activator, a deoxynucleoside triphosphate (dNTP), a ribonucleoside tri-phosphate (rNTP), a single-stranded DNA binding (SSB) protein, Rnase inhibitor, water, a polymerase, reverse transcriptase mix, or a combination thereof that is suitable for an amplification reaction.
  • dNTP deoxynucleoside triphosphate
  • rNTP ribonucleoside tri-phosphate
  • SSB single-stranded DNA binding
  • Non-limiting examples of amplification reactions are transcription mediated amplification (TMA), helicase dependent amplification (HDA), or circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer- dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), and improved multiple displacement amplification (IMDA).
  • TMA transcription mediated amplification
  • HDA helicase dependent amplification
  • cHDA circular helicase dependent amplification
  • SDA strand displacement amplification
  • RPA re
  • solutions, compositions, systems and methods comprise 0.01 ⁇ L, 0.02 ⁇ L, 0.03 ⁇ L, 0.04 ⁇ L, 0.05 ⁇ L, 0.06 ⁇ L, 0.07 ⁇ L, 0.08 ⁇ L, 0.09 ⁇ L, 0.1 ⁇ L, 0.2 ⁇ L, 0.3 ⁇ L, 0.4 ⁇ L, 0.5 ⁇ L, 0.6 ⁇ L, 0.7 ⁇ L, 0.8 ⁇ L, 0.9 ⁇ L, 1 ⁇ L, 2 ⁇ L, 3 ⁇ L, 4 ⁇ L, 5 ⁇ L, 6 ⁇ L, 7 ⁇ L, 8 ⁇ L, 9 ⁇ L, 10 ⁇ L, 20 ⁇ L, 30 ⁇ L, 40 ⁇ L, 50 ⁇ L, 60 ⁇
  • solutions, compositions, systems and methods comprise 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or more of each amplification reagent as described herein.
  • solutions, compositions, systems and methods comprise 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 60 ⁇ M, 70 ⁇ M, 80 ⁇ M, 90 ⁇ M, 100 ⁇ M, 150 ⁇ M, 200 ⁇ M, 250 ⁇ M, 300 ⁇ M, 350 ⁇ M, 400 ⁇ M, 450 ⁇ M, 500 ⁇ M, or more of each amplification reagent as described herein.
  • solutions, compositions, systems and methods comprise 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or more of each amplification reagent as described herein.
  • systems described herein comprise a PCR tube, a PCR well or a PCR plate.
  • the wells of the PCR plate are pre-aliquoted with the reagent for amplifying a nucleic acid, as well as a guide nucleic acid, an effector protein, a multimeric complex, or any combination thereof.
  • the wells of the PCR plate are pre-aliquoted with a guide nucleic acid targeting a target sequence, an effector protein is activated when complexed with the guide nucleic acid and the target sequence, an effector protein is activated when complexed with the guide nucleic acid and the target sequence, and at least one population of a single stranded reporter nucleic acid comprising a detection moiety.
  • a user thus adds the biological sample of interest to a well of the pre-aliquoted PCR plate and measure for the detectable signal with a fluorescent light reader or a visible light reader.
  • systems comprise a PCR plate; a guide nucleic acid targeting a target sequence; an effector protein is activated when complexed with the guide nucleic acid and the target sequence; and a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid is cleaved by the activated nuclease, thereby generating a detectable signal.
  • systems described herein comprise a support medium; a guide nucleic acid targeting a target sequence; and an effector protein is activated when complexed with the guide nucleic acid and the target sequence.
  • nucleic acid amplification is performed in a nucleic acid amplification region on the support medium.
  • the nucleic acid amplification is performed in a reagent chamber, and the resulting sample is applied to the support medium.
  • a system described herein for editing a target nucleic acid comprises a PCR plate; a guide nucleic acid targeting a target sequence; and an effector protein is activated when complexed with the guide nucleic acid and the target sequence.
  • the wells of the PCR plate are pre-aliquoted with the guide nucleic acid targeting a target sequence, and an effector protein is activated when complexed with the guide nucleic acid and the target sequence.
  • a user thus adds the biological sample of interest to a well of the pre-aliquoted PCR plate.
  • the nucleic acid amplification is performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes, or any value 1 to 60 minutes.
  • the amplification reaction is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes.
  • the amplification reaction is performed at a temperature of around 20-45oC.
  • the amplification reaction is performed at a temperature no greater than 20oC, 25oC, 27oC, 30oC, 35oC, 37oC, 40oC, 45oC, 47oC, 50oC, 55oC, 57oC, 60oC, 65oC, 67oC, 70oC, 75oC, 77oC, 80oC, or any value 20 oC to 80 oC.
  • the amplification reaction is performed at a temperature of at least 20oC, 25oC, 27oC, 30oC, 35oC, 37oC, 40oC, 45oC, 47oC, 50oC, 55oC, 57oC, 60oC, 65oC, 67oC, 70oC, 75oC, 77oC, 80oC or any value 20 oC to 80 oC.
  • the amplification reaction is performed at a temperature of 20oC to 45oC, 25oC to 40oC, 30oC to 40oC, 35oC to 40oC, 40oC to 45oC, 45oC to 50oC, 50oC to 55oC, 55oC to 60oC, 35oC to 40oC, 50oC to 65oC, 65oC to 70oC, 70oC to 80oC, 75oC to 80oC.
  • systems comprise primers for amplifying a target nucleic acid to produce an amplification product comprising the target nucleic acid and a PAM.
  • At least one of the primers comprise the PAM that is incorporated into the amplification product during amplification.
  • the compositions for amplification of target nucleic acids and methods of use thereof, as described herein, are compatible with any of the methods disclosed herein including methods of assaying for at least one base difference (e.g., assaying for a SNP or a base mutation) in a target nucleic acid, methods of assaying for a target nucleic acid that lacks a PAM by amplifying the target nucleic acid to introduce a PAM, and compositions used in introducing a PAM by amplification into the target nucleic acid.
  • systems include a package, carrier, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, test wells, bottles, vials, syringes, and test tubes.
  • the containers are formed from a variety of materials such as glass, plastic, or polymers.
  • the system or systems described herein contain packaging materials. Examples of packaging materials include, but are not limited to, pouches, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for intended mode of use.
  • systems described herein include labels listing contents and/or instructions for use, or package inserts with instructions for use.
  • the systems include a set of instructions and/or a label is on or associated with the container.
  • the label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container (e.g., as a package insert).
  • the label is used to indicate that the contents are to be used for a specific therapeutic application.
  • the label indicates directions for use of the contents, such as in the methods described herein.
  • the product after packaging the formed product and wrapping or boxing to maintain a sterile barrier, the product is terminally sterilized by heat sterilization, gas sterilization, gamma irradiation, or by electron beam sterilization. Alternatively, in some embodiments, the product is prepared and packaged by aseptic processing.
  • systems comprise a solid support.
  • an RNP or effector protein is attached to a solid support.
  • the solid support comprises an electrode or a bead.
  • the bead comprises a magnetic bead. Upon cleavage, the RNP is liberated from the solid support and interacts with other mixtures.
  • the effector protein of the RNP flows through a chamber into a mixture comprising a substrate.
  • a reaction occurs, such as a colorimetric reaction, which is then detected.
  • the protein is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme flows through a chamber into a mixture comprising the enzyme.
  • a reaction occurs, such as a calorimetric reaction, which is then detected.
  • systems and methods are employed under certain conditions that enhance an activity of the effector protein relative to alternative conditions, as measured by a detectable signal released from cleavage of a reporter in the presence of the target nucleic acid.
  • the detectable signal is generated at about the rate of trans cleavage of a reporter nucleic acid.
  • the reporter nucleic acid is a homopolymeric reporter nucleic acid comprising 5 to 20 consecutive adenines (SEQ ID NO: 811), 5 to 20 consecutive thymines (SEQ ID NO: 812), 5 to 20 consecutive cytosines (SEQ ID NO: 813), or 5 to 20 consecutive guanines (SEQ ID NO: 814).
  • the reporter is an RNA-FQ reporter.
  • effector proteins disclosed herein recognize, bind, or are activated by, different target nucleic acids having different sequences, but are active toward the same reporter nucleic acid, allowing for facile multiplexing in a single assay having a single ssRNA-FQ reporter.
  • systems are employed under certain conditions that enhance trans cleavage activity of an effector protein.
  • trans cleavage occurs at a rate of at least 0.005 mmol/min, at least 0.01 mmol/min, at least 0.05 mmol/min, at least 0.1 mmol/min, at least 0.2 mmol/min, at least 0.5 mmol/min, or at least 1 mmol/min.
  • systems and methods are employed under certain conditions that enhance cis cleavage activity of the effector protein.
  • Certain conditions that may enhance the activity of an effector protein include a certain salt presence or salt concentration of the solution in which the activity occurs. For example, in some embodiments, cis cleavage activity of an effector protein is inhibited or halted by a high salt concentration.
  • the salt comprises a magnesium salt, a zinc salt, a potassium salt, a calcium salt, a lithium salt, an ammonium salt, or a sodium salt.
  • the salt is magnesium acetate.
  • the salt is magnesium chloride.
  • the salt is potassium acetate.
  • the salt is potassium nitrate.
  • the salt is zinc chloride.
  • the salt is sodium chloride.
  • the salt is potassium chloride.
  • the salt is lithium acetate.
  • the salt is ammonium sulfate.
  • the salt concentration is less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM. In some embodiments, the salt concentration is more than 1 mM, but less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM. In some embodiments, the salt concentration is more than 10 mM, but less than 150 mM, less than 125 mM, less than 100 mM, less than 75 mM, less than 50 mM, or less than 25 mM.
  • the salt is potassium acetate or sodium chloride and the concentration of salt in the solution is about 200 mM. In some embodiments, the salt is potassium acetate or, sodium chloride, lithium acetate, or ammonium sulfate and the concentration of salt in the solution is about 100 mM to about 200 mM.
  • Certain conditions that may enhance the activity of an effector protein include the pH of a solution in which the activity. For example, in some embodiments, increasing pH enhances trans cleavage activity. For example, in some embodiments, the rate of trans cleavage activity increases with increase in pH up to pH 9.
  • the pH is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.
  • the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5.
  • the pH is less than 7.
  • the pH is greater than 7.
  • Certain conditions that may enhance the activity of an effector protein includes the temperature at which the activity is performed. In some embodiments, the temperature is about 25oC to about 80oC.
  • the temperature is about 20°C to about 40°C, about 30°C to about 50°C, or about 40°C to about 60°C. In some embodiments, the temperature is about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55oC, about 60oC, about 65°C, about 70°C, about 75°C, or about 80°C.
  • Such systems may comprise, as described herein, one or more components having any one of the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) or a nucleic acid comprising a nucleotide sequence encoding same.
  • such systems comprise, as described herein, one or more components having a guide nucleic acid or a nucleic acid comprising a nucleotide sequence encoding same.
  • systems comprise one or more components having a guide nucleic acid and an additional nucleic acid. Systems and components thereof may be used to introduce the polypeptides, guide nucleic acids, or combinations thereof into a target cell. Such methods may be used to modify or edit a target nucleic acid.
  • systems comprise the polypeptide, one or more guide nucleic acids, and a reagent for facilitating the introduction of the polypeptide and the one or more guide nucleic acids.
  • system components for the methods comprise a solution, a buffer, a reagent for facilitating the introduction of the polypeptide and the one or more guide nucleic acids, or combinations thereof.
  • a guide nucleic acid (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a polypeptide (e.g., effector protein, effector partner, fusion protein, or combination thereof) (or a nucleic acid comprising a nucleotide sequence encoding same) described herein may be introduced into a host cell by any of a variety of well-known methods.
  • the guide nucleic acid and/or polypeptide may be combined with a lipid.
  • the guide nucleic acid and/or polypeptide may be combined with a particle or formulated into a particle.
  • a host may be any suitable host.
  • a host comprises a host cell.
  • a host cell comprises an in vivo or in vitro eukaryotic cell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity.
  • eukaryotic or prokaryotic cells are, or have been, used as recipients for methods of introduction described herein.
  • eukaryotic or prokaryotic cells comprise the progeny of the original cell which has been transformed by the methods of introduction described herein. It is understood that the progeny of a single cell is not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • a host cell comprises a recombinant host cell or a genetically modified host cell, if a heterologous nucleic acid, e.g., an expression vector, has been introduced into the cell.
  • Methods of introducing a nucleic acid and/or protein into a host cell are known in the art, and any convenient method may be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., a human cell, and the like).
  • a subject nucleic acid e.g., an expression construct/vector
  • a target cell e.g., a human cell, and the like.
  • Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.
  • PKI polyethyleneimine
  • the nucleic acid and/or protein(s) are introduced into a disease cell comprised in a pharmaceutical composition comprising the guide nucleic acid, the polypeptide, a pharmaceutically acceptable excipient, or combinations thereof.
  • molecules of interest such as nucleic acids of interest, are introduced to a host.
  • polypeptides are introduced to a host.
  • vectors such as lipid particles and/or viral vectors are introduced to a host.
  • introduction is for contact with a host or for assimilation into the host, for example, introduction into a host cell.
  • nucleic acids such as a nucleic acid encoding a polypeptide, a nucleic acid that, when transcribed, produces an engineered guide nucleic acid, or combinations thereof, into a host cell.
  • Any suitable method may be used to introduce a nucleic acid into a cell. Suitable methods include, for example, viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.
  • PEI polyethyleneimine
  • introducing one or more nucleic acids into a host cell occurs in any culture media and under any culture conditions that promote the survival of the cells. In some embodiments, introducing one or more nucleic acids into a host cell is carried out in vivo or ex vivo. In some embodiments, introducing one or more nucleic acids into a host cell is carried out in vitro.
  • polypeptides e.g., effector proteins, effector partners, fusion proteins, or combination thereof
  • the RNA is provided by direct chemical synthesis or is transcribed in vitro from a DNA (e.g., encoding the polypeptide).
  • the RNA is introduced into a cell by way of any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).
  • introduction of one or more nucleic acid is through the use of a vector and/or a vector system, accordingly, in some embodiments, compositions and system described herein comprise a vector and/or a vector system.
  • vectors are introduced directly to a host.
  • host cells are contacted with one or more vectors as described herein, and in some embodiments, said vectors are taken up by the cells.
  • Methods for contacting cells with vectors include but are not limited to electroporation, calcium chloride transfection, microinjection, lipofection, micro-injection, contact with the cell or particle that comprises a molecule of interest, or a package of cells or particles that comprise molecules of interest.
  • components described herein are introduced directly to a host.
  • an engineered guide nucleic acid is introduced to a host, specifically introduced into a host cell.
  • Methods of introducing nucleic acids, such as RNA into cells include, but are not limited to direct injection, transfection, or any other method used for the introduction of nucleic acids.
  • polypeptides e.g., effector proteins, effector partners, fusion proteins, or combination thereof
  • polypeptides described herein are introduced directly to a host.
  • polypeptides described herein are modified to promote introduction to a host.
  • polypeptides described herein are modified to increase the solubility of the polypeptide.
  • the polypeptide is optionally fused to a polypeptide domain that increases solubility.
  • the domain is linked to the polypeptide through a defined protease cleavage site, such as TEV sequence which is cleaved by TEV protease.
  • the linker comprises one or more flexible sequences, e.g., from 1 to 10 glycine residues.
  • the cleavage of the polypeptide is performed in a buffer that maintains solubility of the product, e.g., in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like.
  • Domains of interest include endosomolytic domains, e.g., influenza HA domain; and other polypeptides that aid in production, e.g., IF2 domain, GST domain, GRPE domain, and the like.
  • the polypeptide is modified to improve stability.
  • polypeptides is PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.
  • polypeptides are modified to promote uptake by a host, such as a host cell.
  • a polypeptide described herein is fused to a polypeptide permeant domain to promote uptake by a host cell. Any suitable permeant domains may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers.
  • Examples include penetratin, a permeant peptide that is derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia; the HIV-1 tat basic region amino acid sequence, e.g., amino acids 49-57 of a naturally-occurring tat protein; and poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nonaarginine, octa-arginine, and the like.
  • the site at which the fusion is made is selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide.
  • the optimal site is determined by suitable methods.
  • Formulations for introducing systems and compositions to a host Described herein are formulations of introducing compositions or components of a system described herein to a host.
  • such formulations, systems and compositions described herein comprise polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and a carrier (e.g., excipient, diluent, vehicle, or filling agent).
  • a carrier e.g., excipient, diluent, vehicle, or filling agent.
  • the polypeptides are provided in a pharmaceutical composition comprising the polypeptides and any pharmaceutically acceptable excipient, carrier, or diluent.
  • compositions, methods, and systems for modifying e.g., editing
  • modifying refers to changing the physical composition of a target nucleic acid.
  • compositions, methods, and systems disclosed herein modify target nucleic acids, such as making epigenetic modifications of target nucleic acids, which does not change the nucleotide sequence of the target nucleic acids per se.
  • polypeptides e.g., effector proteins, effector partners, fusion proteins, or combination thereof
  • compositions and systems described herein are used for modifying a target nucleic acid, which includes editing a target nucleic acid sequence.
  • modifying a target nucleic acid comprises one or more of: cleaving the target nucleic acid, deleting one or more nucleotides of the target nucleic acid, inserting one or more nucleotides into the target nucleic acid, mutating one or more nucleotides of the target nucleic acid, or otherwise changing one or more nucleotides of the target nucleic acid.
  • modifying a target nucleic acid comprises one or more of: methylating, demethylating, deaminating, or oxidizing one or more nucleotides of the target nucleic acid.
  • compositions, methods, and systems described herein modify a coding portion of a gene, a non-coding portion of a gene, or a combination thereof.
  • modifying at least one gene using the compositions, methods or systems described herein reduce or increase expression of one or more genes.
  • the compositions, methods or systems reduce expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the compositions, methods or systems remove all expression of a gene, also referred to as genetic knock out.
  • compositions, methods or systems increase expression of one or more genes by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.
  • the compositions, methods or systems comprise a nucleic acid expression vector, or use thereof, to introduce polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), guide nucleic acid, donor template or any combination thereof to a cell.
  • the nucleic acid expression vector is a viral vector.
  • Viral vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.
  • the viral vector is a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects.
  • the viral vector is an adeno associated viral (AAV) vector.
  • the nucleic acid expression vector is a non-viral vector.
  • compositions and methods comprise a lipid, polymer, nanoparticle, or a combination thereof, or use thereof, to introduce the polypeptide, guide nucleic acid, donor template or any combination thereof to a cell.
  • lipids and polymers are cationic polymers, cationic lipids, or bio-responsive polymers.
  • the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.
  • methods of modifying comprise contacting a target nucleic acid with one or more components, compositions or systems described herein.
  • a method of modifying comprises contacting a target nucleic acid with at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids.
  • a polypeptides e.g., effector proteins, effector partners, fusion proteins, or combination thereof
  • guide nucleic acids e.g., guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids.
  • a method of modifying comprises contacting a target nucleic acid with a system described herein wherein the system comprises components comprising at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids.
  • polypeptides e.g., effector proteins, effector partners, fusion proteins, or combination thereof
  • a method of modifying comprises contacting a target nucleic acid with a composition described herein comprising at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids; in a composition.
  • a method of modifying as described herein produces a modified target nucleic acid.
  • editing a target nucleic acid sequence introduces a mutation (e.g., point mutations, deletions) in a target nucleic acid relative to a corresponding wildtype nucleotide sequence.
  • editing removes or corrects a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence.
  • editing a target nucleic acid sequence removes/corrects point mutations, deletions, null mutations, or tissue- specific mutations in a target nucleic acid.
  • editing a target nucleic acid sequence is used for generating gene knock-out, gene knock-in, gene editing, gene tagging, or a combination thereof.
  • modifying comprises single stranded cleavage, double stranded cleavage, donor nucleic acid insertion, epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof.
  • cleavage single- stranded or double-stranded is site-specific, meaning cleavage occurs at a specific site in the target nucleic acid, often within the region of the target nucleic acid that hybridizes with the guide nucleic acid spacer sequence.
  • the polypeptides introduce a single-stranded break in a target nucleic acid to produce a cleaved nucleic acid.
  • the polypeptide introduces a break in a single stranded RNA (ssRNA).
  • the polypeptide is coupled to a guide nucleic acid that targets a particular region of interest in the ssRNA.
  • the target nucleic acid, and the resulting cleaved nucleic acid is contacted with a nucleic acid for homologous recombination (e.g., homology directed repair (HDR)) or non-homologous end joining (NHEJ).
  • a double-stranded break in the target nucleic acid is repaired (e.g., by NHEJ or HDR) without insertion of a donor template, such that the repair results in an indel in the target nucleic acid at or near the site of the double-stranded break.
  • an indel is a type of genetic mutation that results from the insertion and/or deletion of one or more nucleotide in a target nucleic acid.
  • an indel varies in length (e.g., 1 to 1,000 nucleotides in length) and be detected using methods well known in the art, including sequencing. If the number of nucleotides in the insertion/deletion is not divisible by three, and it occurs in a protein coding region, it is also a frameshift mutation.
  • Indel percentage is the percentage of sequencing reads that show at least one nucleotide has been mutation that results from the insertion and/or deletion of nucleotides regardless of the size of insertion or deletion, or number of nucleotides mutated. For example, if there is at least one nucleotide deletion detected in a given target nucleic acid, it counts towards the percent indel value. As another example, if one copy of the target nucleic acid has one nucleotide deleted, and another copy of the target nucleic acid has 10 nucleotides deleted, they are counted the same. This number reflects the percentage of target nucleic acids that are edited by a given polypeptide.
  • methods of modifying described herein cleave a target nucleic acid at one or more locations to generate a cleaved target nucleic acid.
  • the cleaved target nucleic acid undergoes recombination (e.g., NHEJ or HDR).
  • cleavage in the target nucleic acid is repaired (e.g., by NHEJ or HDR) without insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site.
  • cleavage in the target nucleic acid is repaired (e.g., by NHEJ or HDR) with insertion of a donor nucleic acid, such that the repair results in an indel in the target nucleic acid at or near the site of the cleavage site.
  • a wild-type reading frame comprises a reading frame that produces at least a partially, or fully, functional protein.
  • a non-wild-type reading frame comprises a reading frame that produces a non-functional or partially non-functional protein.
  • compositions, systems, and methods described herein edit 1 to 1,000 nucleotides or any integer in between, in a target nucleic acid.
  • 1 to 1,000, 2 to 900, 3 to 800, 4 to 700, 5 to 600, 6 to 500, 7 to 400, 8 to 300, 9 to 200, or 10 to 100 nucleotides, or any integer in between are edited by the compositions, systems, and methods described herein.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides are edited by the compositions, systems, and methods described herein.
  • 10, 20, 30, 40, 50, 60, 70, 8090, 100 or more nucleotides, or any integer in between, are edited by the compositions, systems, and methods described herein.
  • 100, 200, 300, 400, 500, 600, 700, 800, 900 or more nucleotides, or any integer in between, are edited by the compositions, systems, and methods described herein.
  • methods comprise use of two or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof).
  • An illustrative method for introducing a break in a target nucleic acid comprises contacting the target nucleic acid with: (a) a first engineered guide nucleic acid comprising a region that binds to a first polypeptide described herein; and (b) a second engineered guide nucleic acid comprising a region that binds to a second polypeptide described herein, wherein the first engineered guide nucleic acid comprises an additional region that hybridizes to the target nucleic acid and wherein the second engineered guide nucleic acid comprises an additional region that hybridizes to the target nucleic acid.
  • the first and second polypeptide are identical. In some embodiments, the first and second polypeptide are not identical.
  • editing a target nucleic acid comprises genome editing.
  • genome editing comprises editing a genome, chromosome, plasmid, or other genetic material of a cell or organism.
  • the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vivo.
  • the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in a cell.
  • the genome, chromosome, plasmid, or other genetic material of the cell or organism is modified in vitro.
  • a plasmid is edited in vitro using a composition described herein and introduced into a cell or organism.
  • editing a target nucleic acid comprises deleting a sequence from a target nucleic acid.
  • a mutated sequence or a sequence associated with a disease is removed from a target nucleic acid.
  • editing a target nucleic acid comprises replacing a sequence in a target nucleic acid with a second nucleotide sequence.
  • a mutated sequence or a sequence associated with a disease is replaced with a second nucleotide sequence lacking the mutation or that is not associated with the disease.
  • editing a target nucleic acid comprises deleting or replacing a sequence comprising markers associated with a disease or disorder.
  • editing a target nucleic acid comprises introducing a sequence into a target nucleic acid.
  • a beneficial sequence or a sequence that reduces or eliminates a disease is inserted into the target nucleic acid.
  • methods comprise inserting a donor nucleic acid into a cleaved target nucleic acid.
  • the donor nucleic acid is inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid.
  • the cleaved target nucleic acid is cleaved at a single location.
  • the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site).
  • the cleaved target nucleic acid is cleaved at two locations.
  • the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites).
  • methods comprise editing a target nucleic acid with two or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof).
  • editing a target nucleic acid comprises introducing a two or more single-stranded breaks in a target nucleic acid.
  • a break is introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid.
  • the guide nucleic acid binds to the effector protein and hybridizes to a region of the target nucleic acid, thereby recruits the effector protein to the region of the target nucleic acid.
  • binding of the effector protein to the guide nucleic acid and the region of the target nucleic acid activate the effector protein, and the activated effector protein introduces a break (e.g., a single stranded break) in the region of the target nucleic acid.
  • editing a target nucleic acid comprises introducing a first break in a first region of the target nucleic acid and a second break in a second region of the target nucleic acid.
  • editing a target nucleic acid comprise contacting a target nucleic acid with a first guide nucleic acid that binds to a first effector protein and hybridizes to a first region of the target nucleic acid and a second guide nucleic acid that binds to a second effector protein or programmable nickase and hybridizes to a second region of the target nucleic acid.
  • the first effector protein introduces a first break in a first strand at the first region of the target nucleic acid
  • the second effector protein introduces a second break in a second strand at the second region of the target nucleic acid.
  • a segment of the target nucleic acid between the first break and the second break is removed, thereby editing the target nucleic acid.
  • a segment of the target nucleic acid between the first break and the second break is replaced (e.g., with donor nucleic acid), thereby editing the target nucleic acid.
  • compositions, systems, and/or methods described herein effect one or more indels
  • the impact on the transcription and/or translation of the target nucleic acid is predicted depending on: 1) the amount of indels generated; and 2) the location of the indel on the target nucleic acid.
  • the edit or mutation is a frameshift mutation.
  • a frameshift mutation is not effected, but a splicing disruption mutation and/or sequence skip mutation is effected, such as an exon skip mutation.
  • methods, systems and compositions described herein edit a target nucleic acid wherein such editing is measured by indel activity.
  • Indel activity measures the amount of change in a target nucleic acid (e.g., nucleotide deletion(s) and/or insertion(s)) compared to a target nucleic acid that has not been contacted by a polypeptide described in compositions, systems, and methods described herein.
  • indel activity is detected by next generation sequencing of one or more target loci of a target nucleic acid where indel percentage is calculated as the fraction of sequencing reads containing insertions or deletions relative to an unedited reference sequence.
  • methods, systems, and compositions comprising polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and guide nucleic acid described herein exhibit 0.0001% to 65% or more indel activity upon contact to a target nucleic acid compared to a target nucleic acid non-contacted with compositions, systems, or by methods described herein.
  • methods, systems, and compositions comprising polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and guide nucleic acid described herein exhibit 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more indel activity.
  • editing of a target nucleic acid as described herein effects one or more mutations comprising splicing disruption mutations, frameshift mutations (e.g., 1+ or 2+ frameshift mutation), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof.
  • the splicing disruption can be an editing that disrupts a splicing of a target nucleic acid or a splicing of a sequence that is transcribed from a target nucleic acid relative to a target nucleic acid without the splicing disruption.
  • the frameshift mutation can be an editing that alters the reading frame of a target nucleic acid relative to a target nucleic acid without the frameshift mutation.
  • the frameshift mutation can be a +2 frameshift mutation, wherein a reading frame is edited by 2 bases.
  • the frameshift mutation can be a +1 frameshift mutation, wherein a reading frame is edited by 1 base.
  • the frameshift mutation is an editing that alters the number of bases in a target nucleic acid so that it is not divisible by three.
  • the frameshift mutation can be an editing that is not a splicing disruption.
  • a sequence as described in reference to the nucleotide sequence deletion, nucleotide sequence skipping, nucleotide sequence reframing, and nucleotide sequence knock-in can be a DNA sequence, a RNA sequence, an edited DNA or RNA sequence, a mutated sequence, a wild-type sequence, a coding sequence, a non-coding sequence, an exonic sequence (exon), an intronic sequence (intron), or any combination thereof.
  • the nucleotide sequence deletion is an editing where one or more sequences in a target nucleic acid are deleted relative to a target nucleic acid without the nucleotide sequence deletion.
  • the nucleotide sequence deletion can result in or effect a splicing disruption or a frameshift mutation.
  • the nucleotide sequence deletion result in or effect a splicing disruption.
  • the nucleotide sequence skipping is an editing where one or more sequences in a target nucleic acid are skipped upon transcription or translation of the target nucleic acid relative to a target nucleic acid without the nucleotide sequence skipping.
  • the nucleotide sequence skipping can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the nucleotide sequence skipping can result in or effect a splicing disruption. In some embodiments, the nucleotide sequence reframing is an editing where one or more bases in a target are edited so that the reading frame of the nucleotide sequence is reframed relative to a target nucleic acid without the nucleotide sequence reframing. In some embodiments, the nucleotide sequence reframing can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the nucleotide sequence reframing can result in or effect a frameshift mutation.
  • the nucleotide sequence knock-in is an editing where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the nucleotide sequence knock-in.
  • the nucleotide sequence knock-in can result in or effect a splicing disruption or a frameshift mutation. In some embodiments, the nucleotide sequence knock-in can result in or effect a splicing disruption.
  • editing of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising splicing disruption mutations, frameshift mutations, sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof.
  • editing of a specific locus can affect any one of a splicing disruption, frameshift (e.g., 1+ or 2+ frameshift), sequence deletion, sequence skipping, sequence reframing, sequence knock-in, or any combination thereof.
  • editing of a target nucleic acid can be locus specific, modification specific, or both.
  • editing of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) described herein and a guide nucleic acid described herein.
  • methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed in vivo.
  • methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed in vitro.
  • a plasmid is edited in vitro using a composition described herein and introduced into a cell or organism.
  • methods of editing a target nucleic acid or modulating the expression of a target nucleic acid is performed ex vivo.
  • methods comprise obtaining a cell from a subject, editing a target nucleic acid in the cell with methods described herein, and returning the cell to the subject.
  • methods of modifying described herein comprise contacting a target nucleic acid with one or more components, compositions or systems described herein.
  • the one or more components, compositions or systems described herein comprise at least one of: a) one or more polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof), or one or more nucleic acids encoding the one or more polypeptides; and b) one or more guide nucleic acids, or one or more nucleic acids encoding the one or more guide nucleic acids.
  • the one or more effector proteins introduce a single-stranded break or a double- stranded break in the target nucleic acid.
  • methods of modifying described herein produce a modified target nucleic acid comprising an engineered nucleic acid sequence that expresses polypeptide having new activity as compared to an unmodified target nucleic acid, or alters expression of an endogenous polypeptide as compared to an unmodified target nucleic acid.
  • methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at a single location.
  • the methods comprise contacting an RNP comprising polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) and a guide nucleic acid to the target nucleic acid.
  • the methods introduce a mutation (e.g., point mutations, deletions) in the target nucleic acid relative to a corresponding wildtype nucleotide sequence.
  • the methods remove or correct a disease-causing mutation in a nucleic acid sequence to produce a corresponding wildtype nucleotide sequence.
  • the methods remove/correct point mutations, deletions, null mutations, or tissue-specific mutations in a target nucleic acid.
  • the methods introduce a single stranded cleavage, a nick, a deletion of one or two nucleotides, an insertion of one or two nucleotides, a substitution of one or two nucleotides, an epigenetic modification (e.g., methylation, demethylation, acetylation, or deacetylation), or a combination thereof to the target nucleic acid.
  • an epigenetic modification e.g., methylation, demethylation, acetylation, or deacetylation
  • the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid.
  • methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid.
  • methods of modifying described herein comprise using one or more guide nucleic acids or uses thereof, wherein the methods modify a target nucleic acid at two different locations.
  • the methods introduce two cleavage sites in the target nucleic acid, wherein a first cleavage site and a second cleavage site comprise one or more nucleotides therebetween.
  • the methods cause deletion of the one or more nucleotides.
  • the deletion restores a wild-type reading frame.
  • the wild-type reading frame produces at least a partially functional protein.
  • the deletion causes a non-wild-type reading frame.
  • a non-wild-type reading frame produces a partially functional protein or non-functional protein.
  • the at least partially functional protein has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 180%, at least 200%, at least 300%, at least 400% activity compared to a corresponding wildtype protein.
  • the methods comprise using an effector protein and two guide nucleic acids, wherein two RNPs cleave the target nucleic acid at different locations, wherein a first RNP comprises the effector protein and a first guide nucleic acid, and wherein a second RNP comprises the effector protein and a second guide nucleic acid.
  • methods comprising using two effector protein and two guide nucleic acids, wherein both RNPs cleave the target nucleic acid at the same location, wherein a first RNP comprises a first effector protein and a first target nucleic acid, and wherein a second RNP comprises a second effector protein and a second target nucleic acid.
  • methods of editing described herein comprise inserting a donor nucleic acid into a cleaved target nucleic acid.
  • the cleaved target nucleic acid formed by introducing a single-stranded break into a target nucleic acid.
  • the donor nucleic acid is inserted at a specified (e.g., effector protein targeted) point within the target nucleic acid.
  • the cleaved target nucleic acid is cleaved at a single location.
  • the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., at a cleavage site).
  • the cleaved target nucleic acid is cleaved at two locations.
  • the methods comprise contacting a target nucleic acid with an effector protein described herein, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein described herein, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site, optionally through NHEJ or single-strand annealing, thereby resulting in the excision of a portion of the target nucleic acid between the first and second cleavage sites from the target nucleic acid; and contacting the target nucleic acid with a donor nucleic acid for homologous recombination, optionally by HDR or NHEJ, thereby introducing a new sequence into the target nucleic acid (e.g., in between two cleavage sites).
  • compositions, methods, and systems described herein modify any one of target nucleic acids described herein.
  • the target nucleic acid comprises a human ALB gene.
  • the methods of modifying a human ALB gene comprise contacting the human ALB gene with one or more components of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • the methods of modifying a human ALB gene comprise contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • compositions, methods, and systems described herein insert a donor nucleic acid in any one of target nucleic acids described herein.
  • the target nucleic acid comprises a human ALB gene.
  • the methods for inserting a donor nucleic acid in a human ALB gene comprise contacting the human ALB gene with one or more components of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • the methods for inserting a donor nucleic acid in a human ALB gene comprise contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • the systems, the compositions and the pharmaceutical compositions comprise the donor nucleic acid.
  • the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of a functional human protein, wherein the human protein comprises an amino acid sequence that is associated with any of diseases recited in TABLE 12.
  • compositions, methods, and systems described herein modify any one of target nucleic acids described herein.
  • the target nucleic acid comprises a human PCSK9 gene.
  • the methods of modifying a human PCSK9 gene comprise contacting the human PCSK9 gene with one or more components of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • the methods of modifying a human PCSK9 gene comprise contacting the human PCSK9 gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein, one or more compositions described herein, or one or more pharmaceutical compositions described herein.
  • Genetically Modified Cells and Organisms [0420] In some embodiments, methods of editing described herein is employed to generate a genetically modified cell.
  • the cell is a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., an archaeal cell).
  • the cell is derived from a multicellular organism and cultured as a unicellular entity.
  • the cell comprises a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation.
  • the cell is progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell.
  • the genetically modified cell comprises a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived.
  • methods of editing described herein is performed in a cell.
  • the cell is in vivo.
  • the cell is ex vivo.
  • the cell is an isolated cell. In some embodiments, the cell is inside of an organism. In some embodiments, the cell is an organism. In some embodiments, the cell is in a cell culture. In some embodiments, the cell is one of a collection of cells. In some embodiments, the cell is a mammalian cell or derived there from. In some embodiments, the cell is a rodent cell or derived there from. In some embodiments, the cell is a human cell or derived there from. In some embodiments, the cell is a eukaryotic cell or derived there from. In some embodiments, the cell is a progenitor cell or derived there from. In some embodiments, the cell is a pluripotent stem cell or derived there from.
  • the cell is an animal cell or derived there from. In some embodiments, the cell is an invertebrate cell or derived there from. In some embodiments, the cell is a vertebrate cell or derived there from. In some embodiments, the cell is from a specific organ or tissue. In some embodiments, the cell is a hepatocyte. In some embodiments, the tissue is a subject’s blood, bone marrow, or cord blood. In some embodiments, the tissue is a heterologous donor blood, cord blood, or bone marrow. In some embodiments, the tissue is an allogenic blood, cord blood, or bone marrow. In some embodiments, the tissue comprises a muscle. In some embodiments, the muscle comprises a skeletal muscle.
  • methods of editing described herein comprise contacting cells with compositions or systems described herein.
  • the methods comprise contacting the cells with a nucleic acid encoding the effector protein at a dose of 3 ⁇ g to 10 ⁇ g (e.g., 3 ⁇ g, 6 ⁇ g, 9 ⁇ g or 10 ⁇ g), and a nucleic acid encoding the guide nucleic acid at a dose of 500 ⁇ M.
  • the contacting comprises electroporation, acoustic poration, optoporation, viral vector-based delivery, iTOP, nanoparticle delivery (e.g., lipid or gold nanoparticle delivery), cell- penetrating peptide (CPP) delivery, DNA nanostructure delivery, or any combination thereof.
  • methods of editing described herein are performed in a subject.
  • the methods comprise administering compositions described herein to the subject.
  • the subject is a human.
  • the subject is a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse).
  • the subject is a vertebrate or an invertebrate.
  • the subject is a laboratory animal. In some embodiments, the subject is a patient. In some embodiments, the subject is at risk of developing, suffering from, or displaying symptoms of a disease. In some embodiments, the subject has a mutation associated with a gene described herein. In some embodiments, the subject displays symptoms associated with a mutation of a gene described herein.
  • XV. Methods of Treating a Disease or Disorder Described herein are methods for treating a disease in a subject by contacting a target nucleic acid with a composition or system described herein, wherein the target nucleic acid is associated with a gene or expression of a gene related to the disease.
  • methods comprise treating, preventing, or inhibiting a disease or disorder associated with a mutation or aberrant expression of a gene.
  • methods for treating a disease or disorder comprise methods of editing a nucleic acid described herein.
  • compositions or systems described herein are for use in a method for treating a disease.
  • compositions or systems described herein are for use in the manufacture of a medicament for treating a disease. [0425]
  • methods comprise administration of a composition(s) or component(s) of a system described herein.
  • the composition(s) or component(s) of the system comprises use of a recombinant nucleic acid (DNA or RNA), administered for the purpose to edit a nucleic acid.
  • the composition or component of the system comprises use of a vector to introduce a functional gene or transgene.
  • vectors comprise nonviral vectors, including cationic polymers, cationic lipids, or bio-responsive polymers.
  • the bio-responsive polymer exploits chemical-physical properties of the endosomal environment (e.g., pH) to preferentially release the genetic material in the intracellular space.
  • vectors comprise viral vectors, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses.
  • the vector comprises a replication-defective viral vector, comprising an insertion of a therapeutic gene inserted in genes essential to the lytic cycle, preventing the virus from replicating and exerting cytotoxic effects.
  • the composition(s) comprises pharmaceutical compositions described herein. Methods of gene therapy that are applicable to the compositions and systems described herein are described in more detail in Ingusci et al., “Gene Therapy Tools for Brain Diseases”, Front. Pharmacol. 10:724 (2019), which is hereby incorporated by reference in its entirety.
  • treating, preventing, or inhibiting disease or disorder in a subject comprises contacting a target nucleic acid associated with a particular ailment with a composition described herein.
  • the methods of treating, preventing, or inhibiting a disease or disorder involves removing, editing, modifying, replacing, transposing, or affecting the regulation of a genomic sequence of a patient in need thereof.
  • the methods of treating, preventing, or inhibiting a disease or disorder involves modulating gene expression.
  • the compositions and systems described herein are for use in therapy.
  • the compositions and systems described herein are for use in treating a disease or condition described herein.
  • compositions described herein in the manufacture of a medicament. Also provided is the use of the compositions described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein.
  • the polypeptides (e.g., effector proteins, effector partners, fusion proteins, or combination thereof) described herein are for use in therapy. In some embodiments, the polypeptides described herein are for use in treating a disease or condition described herein. Also provided is the use of the polypeptides described herein in the manufacture of a medicament. Also provided is the use of the polypeptides described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein.
  • the guide nucleic acids described herein are for use in therapy. In some embodiments, the guide nucleic acids described herein are for use in treating a disease or condition described herein. Also provided is the use of the guide nucleic acids described herein in the manufacture of a medicament. Also provided is the use of the guide nucleic acids described herein in the manufacture of a medicament for therapeutic and/or prophylactic treatment of a disease or condition described herein. [0430] Described herein are compositions, systems and methods for treating a disease in a subject by editing a target nucleic acid associated with a gene or expression of a gene related to the disease.
  • the editing comprises knock-out of a gene comprising the target nucleic acid.
  • the compositions, systems and methods comprise LNPs, wherein the LNPs comprise the effector proteins described herein or nucleic acids encoding the effector proteins, the effector partners described herein or nucleic acids encoding the effector partners, the fusion proteins described herein or nucleic acids encoding the fusion proteins, or combinations thereof.
  • the LNPs comprise chemically modified guide nucleic acids.
  • the LNPs described herein are used for delivering the compositions, or one or more components of the systems described herein to a specific organ (e.g., liver).
  • the compositions, systems and methods comprise AAV particles, wherein the AAV particles comprise nucleic acids encoding the effector proteins described herein, the effector partners described herein, the fusion proteins described herein, or combinations thereof.
  • the AAV particles comprise nucleic acids encoding guide nucleic acids described herein.
  • the AAV particles described herein are used for delivering the compositions, or one or more components of the systems described herein to specific cells (e.g., nerve cells or muscle cells).
  • the one or more components comprise the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the systems described herein.
  • methods comprise administering a composition or cell described herein to a subject.
  • the disease comprises a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof.
  • the disease comprises an inherited disorder, also referred to as a genetic disorder.
  • the disease is the result of an infection or associated with an infection.
  • the compositions are pharmaceutical compositions described herein. [0431]
  • the compositions and methods described herein are used for treating, preventing, or inhibiting a disease or syndrome in a subject.
  • the disease is a liver disease, a lung disease, an eye disease, or a muscle disease.
  • treating, preventing, or inhibiting a disease or syndrome comprises inserting a nucleotide sequence encoding a functional human protein into a safe harbor locus. Exemplary amino acid sequences of functional human proteins are listed in TABLE 12. Exemplary diseases and syndromes include but are not limited to the diseases and syndromes listed in TABLE 13.
  • diseases and syndromes include acute coronary syndrome, angina, arteriosclerosis, atherosclerotic cardiovascular disease, cardiovascular disease (CVD), coronary heart disease, diabetes Type I, diabetes Type II, glaucoma, heart failure, hepatic steatosis, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridaemia, non- alcoholic fatty liver disease (NAFLD), optic neuropathy, sitosterolemia, tendon xanthomatosis, vascular inflammation, xanthelasma or a combination thereof, wherein the diseases and syndromes are associated with one or more mutations in PCSK9 gene.
  • CVD cardiovascular disease
  • CVD cardiovascular disease
  • diabetes Type I diabetes Type II
  • glaucoma glaucoma
  • heart failure hepatic steatosis
  • hypercholesterolemia hyperlipidemia
  • hypertension hypertriglyceridaemia
  • non- alcoholic fatty liver disease NAFLD
  • optic neuropathy sitosterolemia
  • a system comprising a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence comprising a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4 and TABLE 5.
  • Embodiment 2 The system of Embodiment 1 further comprising an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences recited in TABLE 1, and wherein the effector protein binds to the guide nucleic acid.
  • Embodiment 2 wherein the effector protein comprises one or more amino acid substitutions.
  • Embodiment 4 The system of Embodiment 3, wherein the one or more amino acid substitutions independently comprise one or more conservative substitutions, one or more non- conservative substitutions, or combinations thereof.
  • Embodiment 5. The system of Embodiment 3 or 4, wherein the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues.
  • Embodiment 6. The system of Embodiment 5, wherein the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • Embodiment 7. The system of Embodiment 6, wherein the positively charged amino acid residue is Arg (R).
  • Embodiment 8 The system of any one of Embodiments 2-7, wherein the effector protein comprises at least one nuclear localization signal.
  • Embodiment 9. The system of any one of Embodiments 2-8, further comprising an effector partner linked to the effector protein.
  • Embodiment 10. The system of Embodiment 9, wherein the effector partner is directly fused to N terminus or C terminus of the effector protein by an amide bond.
  • Embodiment 11 The system of any one of Embodiments 8-10, wherein the at least one nuclear localization signal comprises an amino acid sequence that is identical to any one of nucleotide sequences recited in TABLE 2.
  • Embodiment 13 The system of any one of Embodiments 1-12, wherein the spacer sequence comprises a nucleotide sequence that is at least partially complementary to a target sequence of a target nucleic acid, wherein the target sequence comprises at least 17 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 11 or a reverse complement thereof.
  • Embodiment 14 The system of any one of Embodiments 2-11, wherein the effector protein recognizes a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences recited in TABLE 3.
  • PAM protospacer adjacent motif
  • Embodiment 15 The system of any one of Embodiments 1-14, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 6.
  • Embodiment 16 The system of any one of Embodiments 1-15, wherein the guide nucleic acid comprises an intermediary sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 8.
  • Embodiment 18 The system of any one of Embodiments 1-17, wherein the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 261 or a reverse complement thereof.
  • Embodiment 19 The system of any one of Embodiments 1-17, wherein the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 512 or a reverse complement thereof.
  • Embodiment 20 The system of any one of Embodiments 1-17, wherein the spacer sequence is at least partially complementary to the target sequence, wherein the target sequence comprises at least 17 contiguous nucleotides of SEQ ID NO: 514 or a reverse complement thereof.
  • Embodiment 21 Embodiment 21.
  • Embodiment 22 The system of Embodiment 21, wherein the spacer sequence is at least 90% identical to any one of the nucleotide sequences of SEQ ID NOS: 87-234 and 262-449.
  • Embodiment 23 The system of Embodiment 22, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
  • Embodiment 24 The system of Embodiment 23, wherein the effector protein comprises one or more amino acid substitutions independently selected from I80R, T84R, K105R, G210R, C202R, A218R, D220R, E225R, C246R, Q360R or a combination thereof relative to SEQ ID NO: 1.
  • Embodiment 25 The system of Embodiment 23, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, and wherein the effector protein comprises one or more amino acid substitutions independently selected from K58W, I80K, N193K, S209F, A218K, E225K, N286K, M295W, M298L, A306K, Y315M or a combination thereof relative to SEQ ID NO: 1.
  • Embodiment 26 Embodiment 26.
  • Embodiment 27 The system of any one of Embodiments 23-26, wherein the repeat sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 251.
  • Embodiment 28 The system of any one of Embodiments 23-27, wherein the guide nucleic acid is a crRNA.
  • Embodiment 29 The system of any one of Embodiments 23-27, wherein the guide nucleic acid is a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • Embodiment 30 The system of Embodiment 29, wherein the handle sequence comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences of SEQ ID NO: 257-259.
  • Embodiment 31 The system of any one of Embodiments 23-30, wherein the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 87-234.
  • Embodiment 32 The system of any one of Embodiments 23-30, wherein the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 262-449.
  • Embodiment 33 Embodiment 33.
  • Embodiment 19 or 20 wherein the spacer sequence is at least 90% identical to any one of the nucleotide sequences of SEQ ID NOS: 235-250 and 450-511.
  • Embodiment 34 The system of Embodiment 33, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.
  • the effector protein comprises one or more amino acid substitutions independently selected from L26R, E157A, E164A, E164L, E166A, E166I, E170A, I471T, I489A, I489S, Y490S, Y490A, F491A, F491S, F491G, D495G, D495R, D495K, K496A, K496S, K498A, K498S, K500A, K500S, D501R, D501G, D501K, V502A, V502S, K504A, K504S, S505R, D506A, or a combination thereof relative to SEQ ID NO: 2.
  • Embodiment 36 The system of Embodiment 34, wherein the effector protein comprises one or more amino acid substitutions independently selected from T5R, L26K, L26R, A121Q, S198R, S223P, E258K, I471T, S579R, F701R, or a combination thereof relative to SEQ ID NO: 2.
  • Embodiment 37 The effector protein of Embodiment 34, wherein the effector protein comprises a substitution of L26R, I471T or a combination thereof relative to the amino acid sequence of SEQ ID NO: 2.
  • Embodiment 38 Embodiment 38.
  • Embodiment 39 The system of any one of Embodiments 33-38, wherein the repeat sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 252-254.
  • Embodiment 40 The system of any one of Embodiments 33-39, wherein the guide nucleic acid is a crRNA.
  • Embodiment 41 Embodiment 41.
  • Embodiment 42 The system of any one of Embodiments 33-40, wherein the spacer sequence comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOS: 235-250.
  • Embodiment 42 The system of any one of Embodiments 33-40, wherein the spacer sequence comprises any one of SEQ ID NOS: 450-511.
  • Embodiment 43 The system of any one of Embodiments 33-42, wherein the guide nucleic acid comprises one or more phosphorothioate (PS) backbone modifications, 2’-fluoro (2’-F) sugar modifications, or 2’-O-Methyl (2’OMe) sugar modifications.
  • PS phosphorothioate
  • Embodiment 40 or 41 wherein the guide nucleic acid comprises PS backbone modification between -3 and -2 positions of the guide nucleic acid, wherein the repeat sequence comprises at least 24 nucleotides.
  • Embodiment 45 The system of Embodiment 44, wherein the guide nucleic acid further comprises at least one modification between -16 and -12 positions of the repeat sequence present in the guide nucleic acid.
  • Embodiment 46 The system of Embodiment 45, wherein the at least one modification comprises 2’OMe sugar modification at -14 position of the repeat sequence present in the guide nucleic acid.
  • Embodiment 47 Embodiment 47.
  • Embodiment 45 wherein the at least one modification comprises 2’OMe sugar modification at -16 position of the repeat sequence present in the guide nucleic acid.
  • Embodiment 48 The system of Embodiment 45, wherein the at least one modification comprises PS backbone modification between -13 and -12 positions of the repeat sequence present in the guide nucleic acid.
  • Embodiment 49 The system of Embodiment 45, wherein the at least one modification comprises PS backbone modification between -14 and -13 positions of the repeat sequence present in the guide nucleic acid.
  • Embodiment 50 Embodiment 50.
  • Embodiment 51 A system comprising a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 565.
  • Embodiment 52 The system of Embodiment 51, wherein the system further comprises an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence of SEQ ID NO: 573, and wherein the effector protein binds to the guide nucleic acid.
  • Embodiment 53 The system of any one of Embodiment 1-52, wherein the system further comprises a donor nucleic acid.
  • Embodiment 54 The system of Embodiment 53, wherein the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of a functional human protein, wherein the human protein comprises an amino acid sequence that is associated with any of disease recited in TABLE 12.
  • Embodiment 55 The system of any one of Embodiments 1-54 further comprising a nucleic acid expression vector, wherein the expression vector comprises at least one of the nucleic acid encoding the effector protein; and the nucleic acid encoding the guide nucleic acid.
  • Embodiment 56 The system of Embodiment 55, wherein the nucleic acid expression vector further comprises a nucleotide sequence encoding untranslated regions of any one of the sequences recited in TABLE 20.
  • Embodiment 57 The system of Embodiment 55 or 56, wherein the nucleic acid expression vector further comprises the donor nucleic acid.
  • Embodiment 58 The system of any one of Embodiments 55-57, wherein the nucleic acid expression vector is a viral vector.
  • Embodiment 59 The system of Embodiment 58, wherein the viral vector is an adeno associated viral (AAV) vector.
  • Embodiment 60 The system of Embodiment 55, wherein the nucleic acid expression vector further comprises a nucleotide sequence encoding untranslated regions of any one of the sequences recited in TABLE 20.
  • Embodiment 57 The system of Embodiment 55 or 56, wherein the nucleic acid expression vector further comprises the
  • Embodiment 61 The system of Embodiment 60, the mRNA further comprises one or more of the untranslated regions comprising a nucleotide sequence of any one of the nucleotide sequences recited in TABLE 20.
  • Embodiment 62 The system of any one of Embodiments 1-57 further comprising a lipid or a lipid nanoparticle.
  • Embodiment 63 Embodiment 63.
  • Embodiment 64 A pharmaceutical composition, comprising: the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62 or the composition of Embodiment 63; and a pharmaceutically acceptable excipient.
  • Embodiment 65 A pharmaceutical composition, comprising: the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62 or the composition of Embodiment 63; and a pharmaceutically acceptable excipient.
  • a method of treating a disease associated with a mutation or aberrant expression of a protein in a subject in need thereof comprising administering to the subject the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62, the composition of Embodiment 63, or the pharmaceutical composition of Embodiment 64.
  • Embodiment 66 The method of Embodiment 65, wherein the subject has a genetic disorder.
  • Embodiment 67 The method of Embodiment 66, wherein the subject has one or more mutations.
  • Embodiment 68 Embodiment 68.
  • Embodiment 67 wherein the one or more mutations comprise a point mutation, a single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • Embodiment 69 The method of any one of Embodiments 65-68, wherein the mutation is associated with one or more of protein expression, protein activity, and protein structural stability.
  • Embodiment 70 The method of any one of Embodiments 66-69, wherein the genetic disorder comprises any one of the diseases recited in TABLE 12.
  • Embodiment 71 Embodiment 71.
  • a method of modifying human ALB gene comprising contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62, the composition of Embodiment 63, or the pharmaceutical composition of Embodiment 64.
  • Embodiment 72 A method of modifying human PCSK9 gene, the method comprising contacting the human PCSK9 gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62, the composition of Embodiment 63, or the pharmaceutical composition of Embodiment 64.
  • Embodiment 73 Embodiment 73.
  • a method of inserting a donor nucleic acid in human ALB gene comprising contacting the human ALB gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 53-62, the composition of Embodiment 63, or the pharmaceutical composition of Embodiment 64, wherein the system, the composition and the pharmaceutical composition comprise the donor nucleic acid.
  • Embodiment 74 Embodiment 74.
  • a method of inserting a donor nucleic acid in human PCSK9 gene comprising contacting the human PCSK9 gene with the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 53-62, the composition of Embodiment 63, or the pharmaceutical composition of Embodiment 64, wherein the system, the composition and the pharmaceutical composition comprise the donor nucleic acid.
  • Embodiment 75 The method of Embodiment 73 or 74, wherein the donor nucleic acid comprises a nucleotide sequence encoding an amino acid sequence of a functional human protein, wherein the human protein comprises an amino acid sequence that is associated with any of diseases recited in TABLE 12.
  • Embodiment 76 The method of any one of Embodiments 65-75, wherein the method is performed in a cell.
  • Embodiment 77 The method of any one of Embodiments 65-76, wherein the method is performed in vivo.
  • Embodiment 78 A cell comprising the guide nucleic acid or the nucleic acid encoding the guide nucleic acid of the system of any one of the Embodiments 1-62 or the composition of Embodiment 63.
  • Embodiment 79 A cell that comprises a target nucleic acid modified by the system of any one of Embodiments 1-62 or the composition of Embodiment 63.
  • Embodiment 80 The method of any one of Embodiments 65-75, wherein the method is performed in a cell.
  • Embodiment 77 The method of any one of Embodiments 65-76, wherein the method is performed in vivo.
  • Embodiment 78 A cell comprising the guide nucleic acid or the nucleic
  • Embodiment 78 or 79 wherein the cell is a hepatocyte.
  • Embodiment 81 The cell of any one of Embodiments 78-80, wherein the cell is a mammalian cell.
  • Embodiment 82 The cell of any one of Embodiments 78-81, wherein the cell is a human cell.
  • Embodiment 83 The cell of any one of Embodiments 78-82, wherein the cell is a stem cell, progenitor cell, induced pluripotent stem cell (iPSC) or a cell derived from an iPSC.
  • Embodiment 84 Embodiment 84.
  • TABLE 1 provides illustrative amino acid sequences of effector proteins that are useful in the compositions, systems and methods described herein.
  • TABLE 1. Exemplary Effector Protein Amino Acid Sequence
  • TABLE 1.1 provides illustrative amino acid sequences of effector proteins that are useful in the compositions, systems and methods described herein.
  • TABLE 1.1 Exemplary Effector Protein Variant and Fusion Protein Amino Acid Sequences
  • TABLE 2 provides illustrative sequences of exemplary heterologous polypeptide modifications of effector protein(s) that are useful in the compositions, systems and methods described herein.
  • TABLE 2. Exemplary Nuclear Localization Sequences
  • TABLE 3 provides illustrative PAM sequences that are useful in the compositions, systems and methods described herein.
  • TABLE 4 provides illustrative spacer sequences for use with the compositions, systems and methods of the disclosure.
  • TABLE 4 Exemplary Spacer Sequences targeting human ALB gene
  • TABLE 4.1 provides illustrative spacer sequences for use with the compositions, systems and methods of the disclosure.
  • TABLE 4.1 Exemplary Spacer Sequences targeting human ALB gene [0522]
  • TABLE 5 provides illustrative spacer sequences for use with the compositions, systems and methods of the disclosure.
  • TABLE 5 Exemplary Spacer Sequences targeting PCSK9 gene
  • TABLE 5.1 provides illustrative spacer sequences for use with the compositions, systems and methods of the disclosure.
  • TABLE 6 provides illustrative repeat sequences for use in guide nucleic acids that are useful in the compositions, systems and methods described herein.
  • TABLE 6 Exemplary Repeat Sequences
  • TABLE 7 provides an illustrative linker sequence for use in guide nucleic acids that are useful in the compositions, systems and methods described herein.
  • TABLE 7 Exemplary Linker Sequence
  • TABLE 8 provides an illustrative intermediary sequence for use in guide nucleic acids that are useful in the compositions, systems and methods described herein.
  • TABLE 8 Exemplary Intermediary Sequence [0527]
  • TABLE 9 provides exemplary compositions comprising an effector protein described herein and handle sequences.
  • TABLE 9 Exemplary Handle Sequence [0528]
  • TABLE 10 provides exemplary compositions comprising an effector protein described herein and gRNAs.
  • TABLE 10. Exemplary Compositions of Effector Protein and Guide Nucleic Acids [0529]
  • TABLE 11 provides illustrative target nucleic acid sequences for use with the compositions, systems, and methods of the disclosure. TABLE 11. Target Nucleic Acid Sequences
  • TABLE 12 provides exemplary functional human protein sequences. TABLE 12. Exemplary Functional Human Proteins
  • TABLE 13 provides illustrative diseases and syndromes for compositions, systems and methods described herein. TABLE 13. DISEASES AND SYNDROMES
  • Example 1 Identifying active gRNAs targeting intron 1 of human ALB gene for CasM.265466 effector protein [0533] CasM.265466 system is tested for its indel activity for gRNA against targeted intron 1 of human albumin. Briefly, CasM.265466 system includes composition nos. 1-46 as recited in TABLE 10.
  • CasM.265466 system includes the effector protein CasM.265466 (SEQ ID NO: 1) and sgRNA as described in TABLE 10 are used that are targeting intron 1 of human Albumin gene (SEQ ID NO: 261) in mammalian cell line.
  • SEQ ID NO: 1 the effector protein CasM.265466
  • sgRNA as described in TABLE 10 are used that are targeting intron 1 of human Albumin gene (SEQ ID NO: 261) in mammalian cell line.
  • 20,000 cells are seeded in 96-well plates. After 24 hours, cells are transfected with 100 ng total RNA (1:1 mRNA to gRNA ratio) using Lipofectamine MessengerMax. 48 hours post-transfection, DNA is extracted. DNA editing is quantified by amplicon sequencing.
  • CasPhi.12 system is tested for its indel activity for gRNA against targeted intron 1 of human albumin. Briefly, CasPhi.12 system includes composition no.48 as recited in TABLE 10. As disclosed, CasPhi.12 system includes the effector protein CasPhi.12 (SEQ ID NO: 2) and crRNA as described in TABLE 10 are used that are targeting intron 1 of human Albumin gene (SEQ ID NO: 261) in mammalian cell line.
  • CasM.265466 system includes the effector protein CasM.265466 (SEQ ID NO: 1) and sgRNA as described in TABLE 10 are used that are targeting PCSK9 gene (SEQ ID NO: 512) in mammalian cell line.
  • 20,000 cells are seeded in 96-well plates. After 24 hours, cells are transfected with 100 ng total RNA (1:1 mRNA to gRNA ratio) using Lipofectamine MessengerMax. 48 hours post-transfection, DNA is extracted. DNA editing is quantified by amplicon sequencing.
  • CasPhi.12 system is tested for its indel activity for gRNA against targeted PCSK9 gene. Briefly, CasPhi.12 system includes composition no.49 as recited in TABLE 10. As disclosed, CasPhi.12 system includes the effector protein CasPhi.12 (SEQ ID NO: 2) and crRNA as described in TABLE 10 are used that are targeting PCSK9 gene (SEQ ID NO: 512) in mammalian cell line. [0540] For determining indel activity for CasPhi.12 system using compositions recited in TABLE 10, 20,000 cells are seeded in 96-well plates.
  • CasM.265466 system is tested for its indel activity for gRNA against targeted exon 1-2 of PCSK9 gene. Briefly, CasM.265466 system includes composition no. 47 as recited in TABLE 10.
  • CasM.265466 system includes the effector protein CasM.265466 (SEQ ID NO: 1) and sgRNA as described in TABLE 10 are used that are targeting exon 1-2 of PCSK9 gene (SEQ ID NO: 514) in mammalian cell line.
  • SEQ ID NO: 1 the effector protein CasM.265466
  • sgRNA as described in TABLE 10 are used that are targeting exon 1-2 of PCSK9 gene (SEQ ID NO: 514) in mammalian cell line.
  • 20,000 cells are seeded in 96-well plates. After 24 hours, cells are transfected with 100 ng total RNA (1:1 mRNA to gRNA ratio) using Lipofectamine MessengerMax. 48 hours post-transfection, DNA is extracted. DNA editing is quantified by amplicon sequencing.
  • CasPhi.12 system is tested for its indel activity for gRNA against targeted exon 1-2 of PCSK9 gene. Briefly, CasPhi.12 system includes composition no. 49 as recited in TABLE 10. As disclosed, CasPhi.12 system includes the effector protein CasPhi.12 (SEQ ID NO: 2) and crRNA as described in TABLE 10 are used that are targeting exon 1-2 of PCSK9 gene (SEQ ID NO: 514) in mammalian cell line.
  • 20,000 cells are seeded in 96-well plates. After 24 hours, cells are transfected with 100 ng total RNA (1:1 mRNA to gRNA ratio) using Lipofectamine MessengerMax. 48 hours post-transfection, DNA is extracted. DNA editing is quantified by amplicon sequencing.
  • Example 7 Identifying guide nucleic acids for modifying the human ALB gene with a CasM.265466 effector protein variant
  • D220R variant of CasM.265466 SEQ ID NO: 1
  • various gRNAs (described in TABLE 14) were tested for their ability to integrate a luciferase reporter into intron 1 of a human albumin gene (SEQ ID NO: 261) in primary human hepatocytes (PHH).
  • PHH cells were transfected at 1:1 mRNA to gRNA ratio using Lipofectamine MessengerMax and co-transduced with AAVDJ-nLuc reporter at 1E4 MOI. After 72 hours, luciferase reporter integration was quantified by luciferase assay. SpyCas9 and guide G009874 was used as a positive control.
  • FIGs. 1A-1C show results of the luciferase assay. An analysis of FIGs. 1A-1C indicates that at least 6 of the tested guides (R10250, R10246, R17955, R17915, R17918, R17908 produced more than 60% integration. While data are shown only for gRNAs that demonstrated notable activity in this experiment, activity of additional gRNAs was detected. [0547] CasM.265466 D220R and additional guides (described in TABLE 15) targeting a PAM of 5’- NNTN-3’ in the human albumin intron 1 gene were similarly tested. TABLE 15. gRNA USED FOR TARGETING ALB GENE [0548] FIGs.
  • FIGs. 2A-2C show results of the luciferase assay.
  • An analysis of FIGs. 2A-2C indicates that at least one of the tested guides (R17934) showed similar level of integration (>60%) as R10250. While data are shown only for gRNAs that demonstrated notable activity in this experiment, activity of additional gRNAs was detected.
  • Activity of CasM.265466 D220R, and gRNAs R10250, R17934, R10246, R17955, R17915, R17918 and R17908 were verified in an additional lot of cells from a second donor. See FIG.3.
  • Example 8 Identifying active gRNAs for integrating a luciferase reporter gene within human ALB gene for CasPhi.12 effector protein
  • L26R variant of CasPhi.12 SEQ ID NO: 2
  • various gRNAs (described in TABLE 16) were tested for their ability to integrate a luciferase reporter into intron 1 of a human albumin gene (SEQ ID NO: 261) in primary human hepatocytes (PHH). Briefly, PHH cells were transfected at 1:1 mRNA to gRNA ratio using Lipofectamine MessengerMax and co-transduced with AAVDJ-nLuc reporter at 1E4 MOI. After 72 hours, luciferase reporter integration was quantified by luciferase assay. SpyCas9 and guide G009874 was used as a positive control. TABLE 16.
  • FIG. 4 shows results of the luciferase assay. An analysis of FIG. 4 indicates that at least 5 of the tested guides (R10039, R10035, R10091, R10058, and R10092) produced more than 60% integration. Activity of these guides were verified in an additional lot of cells from a second donor. See FIG.5.
  • Example 9 Exemplary CasPhi.12 variant testing in mice
  • the lipid nanoparticles (LNPs) of the present disclosure were formulated using I471T variant of CasPhi.12 effector protein (SEQ ID NO: 2) and guide RNA (mA*mU*mA*GAUUGCUCCUUACGAGGAGACGAGCAACGGCGGAAmG*mG*mU (SEQ ID NO: 566)) that is complementary to mouse PCSK9 gene.
  • Cas9 mRNA and guide R8217 were injected as a control. The study was repeated, and each study ended 2-7 days post injection. Liver samples were collected for indel analysis by NGS. Data representative of multiple experiments, as provided in FIG.
  • Example 10 In vivo testing of LNP Formulations [0555] Different LNPs and formulations of the present disclosure are tested for in vivo editing in mice with mRNA encoding a variant of CasM.265466 (SEQ ID NO: 1) with a D220R amino acid substitution (D220R effector protein variant) and associated guide nucleic acid targeting a gene of interest.
  • the D220R effector protein variant is modified to comprise NLS sequences at N-terminus (SEQ ID NO: 567) and C-terminus (SEQ ID NO: 4).
  • mRNA encoding the D220R effector variant is represented by the polynucleotide sequence recited in TABLE 17.
  • NLSs and UTRs could be used in this experiment.
  • mice are administered to mice along with an associated mouse guide nucleic acid (mA*mU*mA*GAUUGCUCCUUACGAGGAGACGAGCAACGGCGGAAmG*mG*mU (SEQ ID NO: 566)) intravenously through tail vein at 2 mg/kg dose.
  • Mouse guide nucleic acid mA*mU*mA*GAUUGCUCCUUACGAGGAGACGAGCAACGGCGGAAmG*mG*mU (SEQ ID NO: 566)
  • Blood samples and/or tissue samples e.g., liver samples
  • are harvested at various time points after administration and editing of the target gene is analyzed by NGS.
  • Example 11 Lipid nanoparticle formulations based on CKK-E12 [0557]
  • the lipid nanoparticles (LNPs) of the present disclosure are formulated using mRNA encoding effector protein of any one of SEQ ID NO: 1-2 and a gRNA comprising a spacer sequence that is complementary to a target sequence within any one of the target nucleic acid sequences of TABLE 11.
  • the mRNA and gRNA are mixed and diluted to various concentrations, as shown in TABLE 18 below, in a pH of about 3 to about 5 in acetate buffer or citrate buffer.
  • Cationic lipid, cKK-E12, cholesterol, DMG-PEG 2000 and DOPE (1,2-diolyeyl-sn-glycero-3-phosphoethanolamine) are dissolved in ethanol at various molar ratios.
  • the lipid mixture and RNA mixture are further mixed in a microfluidic device at the various cKK-E12/RNA weight to weight ratios.
  • the formulations are then further dialyzed against a TRIS-saline buffer or phosphate buffered saline (PBS) at a pH of about 7.4 and concentrated to a desired concentration by Amicon filtration.
  • the isolated LNPs are characterized to determine the encapsulation efficiency (EE), polydispersity index (PDI) and average particle size. TABLE 18.
  • Example 12 In vivo testing of LNPs comprising mRNA encoding CasPhi.12 in mice
  • the lipid nanoparticles (LNPs) of the present disclosure are formulated using mRNA encoding effector protein of any one of SEQ ID NO: 1-2, a variant thereof or a fusion protein thereof, and a gRNA comprising a spacer sequence that is complementary to a target sequence within any one of the target nucleic acid recited in TABLE 11.
  • IV intravenous
  • LNP comprising 35 mol% CKK-E12, 16 mol% DOPE, 46.5 mol% cholesterol and 2.5 mol% DMG-PEG2000, with a CKK-E12/RNA w/w 10:1.
  • the study is repeated, and each study ended 2-7 days post injection. Liver samples are collected for indel analysis by NGS. % indel generated is compared to different mRNA lots and formulation runs.
  • Example 13 Lipid Nanoparticle formulations comprising mRNA encoding CasM.265466
  • lipid nanoparticles are formulated using an mRNA having a nucleotide sequence encoding any one of the effector proteins of TABLE 1, a variant thereof or a fusion protein thereof, and two different guide RNAs.
  • Cas9 system is used as a positive control.
  • the guide RNAs each target different portion of the target gene.
  • the mRNA and guide RNAs are mixed and diluted to various concentrations, as shown in TABLE 19 below, at a pH of about 3 to about 5 in acetate buffer or citrate buffer.
  • Ionizable lipids, phospholipids, cholesterol and PEG lipids are dissolved in ethanol at various molar ratios.
  • the lipid mixtures and RNA mixtures are further mixed in a microfluidic device at the various ionizable/RNA weight to weight ratios.
  • the formulations are then further dialyzed against a Tris-saline buffer or phosphate buffered saline (PBS) at a pH of about 7.4 and concentrated to a desired concentration by Amicon filtration.
  • PBS Tris-saline buffer or phosphate buffered saline
  • the isolated LNPs are characterized to determine the encapsulation efficiency (EE), polydispersity index (PDI) and average particle size.
  • Example 14 CasPhi.12 system mediated editing of target nucleic acid using chemically modified guides [0564] Two sets of chemical modifications were designed to identify compatible modifications for a guide nucleic acid in combination with L26R variant of WT CasPhi.12 (SEQ ID NO: 2).
  • the guide comprised a nucleotide sequence of AUAGAUUGCUCCUUACGAGGAGACCUUUGCACUUUCCUUAG (SEQ ID NO: 745) was modified.
  • the two sets included the following modifications: (1) Unbiased tiling of modifications; and (2) Combinatorial modifications rationally designed based on predicted structure of the effector protein.
  • FIGS. 7A-7F show various guide nucleic acid modifications that were tested.
  • the modifications included one or more 2’OMe sugar modifications, and one or more PS backbone modifications.
  • PHA primary human hepatocytes
  • FIGS.8A-8B show results of luciferase assay for unbiased modifications.
  • FIGS. 9A-9C show results of luciferase assay for combinatorial modifications, which are divided into three parts, FIGS.9A-9C, for legibility.
  • Lipid nanoparticles were formulated using variants of WT CasPhi.12 effector protein (SEQ ID NO: 2) and seven guide RNA.
  • Cas9 mRNA and guide were used as a control.
  • 500,000 PHH were transfected with LNPs comprising 2 ⁇ g total RNA (1:1 mRNA/gRNA ratio) at 24-well scale by MessengerMax and co-transduced with 1E4 MOI AAVDJ- nLuc reporter. After 72 hours, integration was quantified by luciferase assay.
  • FIG. 10 shows results of the luciferase assay.
  • CasPhi.12 I471T variant has variable activity across targets, increasing integration for some guides but decreasing for others. Also, CasPhi.12 L26R/I471T variant has improved integration activity across all guides, with nearly 2-fold activity increase for at least one guide, R10039.
  • Example 16 CasPhi.12 variant mediated editing of target nucleic acid
  • Lipid nanoparticles (LNPs) were formulated using variants of WT CasPhi.12 and tested for their capacity to generate indels in PCSK9 gene. The variants included L26R substitution, I471T substitution and both, L26R and I471T, substitutions.
  • R8860 (mA*mU*mA*GAUUGCUCCUUACGAGGAGACGAGCAACGGCGGAAmG*mG*mU (SEQ ID NO: 566)); (2) R8866 (mA*mU*mA*GAUUGCUCCUUACGAGGAGACAUGUCACAGAGUGG mG*mA*mC (SEQ ID NO: 774)); (3) R16962 (auagauugcuccuuacgaggagacAGGUGAGAUCUAGG GAG (SEQ ID NO: 775)); and (4) R17005 (auagauugcuccuuacgaggagacAUCCCUAGAAGCAGCUA (SEQ ID NO: 776)).
  • mice were injected with a single dose (2 mpk) of LNPs comprising mRNA encoding effector protein and the guide RNA (1:3 ratio).
  • Cas9 mRNA and guide were injected as a control. Liver was harvested four days post injection and NGS analysis was performed. The results of the experiment are provided in FIG. 11. An analysis of FIG. 11 indicates that, while editing is relatively low for R16962, the double mutant was able to rescue editing. This suggests that, for some guide/dose regimes, the double mutant may be superior to the single mutant.
  • Example 17 UTRs for gene editing by CasPhi.12 system
  • Activity of systems were assessed in primary human hepatocytes (PHH) comparing ability to integrate a gene using two different UTRs.
  • Nucleotide sequences of the two UTRs are provided in TABLE 20. TABLE 20.
  • Nucleotide sequences of UTRs [0574] Briefly, 500,000 PHH from two different donors were transfected with 2 ⁇ g total RNA (1:1 mRNA/gRNA ratio) at 24-well scale by MessengerMax and co-transduced with 1E4 MOI AAVDJ- nLuc reporter. After 72 hours, integration was quantified by luciferase assay.
  • FIG.12 shows results of the luciferase assay, which is representative of two different donors. [0575] An analysis of FIG.12 indicates that any of the two UTRs can be used for gene integration. [0576] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Abstract

L'invention concerne des compositions, des systèmes et des procédés comprenant des protéines effectrices, et leurs utilisations. Ces protéines effectrices peuvent être caractérisées en tant que protéines associées à CRISPR (Cas). Diverses compositions, systèmes et procédés de la présente divulgation peuvent tirer profit des activités de ces protéines effectrices pour l'édition et/ou l'ingénierie d'acides nucléiques.
PCT/US2024/033100 2023-06-08 2024-06-07 Compositions et procédés pour la modification de gènes humains exprimés par des cellules hépatiques Pending WO2024254519A2 (fr)

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