WO2023220654A2 - Compositions de protéines effectrices et procédés d'utilisation associés - Google Patents

Compositions de protéines effectrices et procédés d'utilisation associés Download PDF

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WO2023220654A2
WO2023220654A2 PCT/US2023/066848 US2023066848W WO2023220654A2 WO 2023220654 A2 WO2023220654 A2 WO 2023220654A2 US 2023066848 W US2023066848 W US 2023066848W WO 2023220654 A2 WO2023220654 A2 WO 2023220654A2
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nucleic acid
sequence
effector protein
seq
amino acid
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WO2023220654A3 (fr
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Sean Chen
Lucas Benjamin HARRINGTON
William Douglass WRIGHT
Aaron DELOUGHERY
Wiputra Jaya HARTONO
Benjamin Julius RAUCH
Stepan TYMOSHENKO
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Mammoth Biosciences, Inc.
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Publication of WO2023220654A2 publication Critical patent/WO2023220654A2/fr
Publication of WO2023220654A3 publication Critical patent/WO2023220654A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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 [CRISPRs]
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • CRISPR/Cas systems Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas proteins), sometimes referred to as a CRISPR/Cas system, were first identified in certain bacterial species and are now understood to form part of a prokaryotic acquired immune system.
  • 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. While CRISPR/Cas proteins are involved in the acquisition, targeting and cleavage of foreign DNA or RNA, the systems may also contain a CRISPR array, which includes direct repeats flanking short spacer sequences that, in part, guide Cas proteins to their targets.
  • CRISPR/Cas systems has revolutionized the field of genomic manipulation and engineering, and therapeutic applications of these systems are being explored.
  • compositions comprising: an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, wherein the target sequence comprises a nucleotide sequence within a human safe harbor locus
  • the donor nucleic acid encodes a transgene that comprises a functional human protein that is expressed upon incorporation into the human safe harbor locus.
  • compositions comprising an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid, 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
  • the effector protein recognizes a target sequence that is in proximity to or adjacent to a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences recited in TABLE 3.
  • compositions comprising: an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, wherein the target sequence comprises a nucleotide sequence within a human safe harbor locus, and wherein the human safe harbor locus is located in human chromosome 2 or human chromosome 4.
  • compositions comprising an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid.
  • 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.
  • compositions comprising an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical to SEQ ID NO: 515 or 516.
  • compositions comprising an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid, wherein the guide nucleic acid comprises a spacer sequence, wherein the spacer sequence is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, 13, 47, and 228-231.
  • the linker sequence comprises a nucleotide sequence that is identical to the nucleotide sequence recited in TABLE 8. In some embodiments, the linker sequence comprises a nucleotide sequence that is identical to SEQ ID NO: 265. In some embodiments, the target sequence comprises a nucleotide sequence within SEQ ID NO: 266. In some embodiments, the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 13 and 231. In some embodiments, the spacer sequence is at least 90% identical to any one of SEQ ID NO: 536-604.
  • compositions described herein wherein the composition comprising a lipid or a lipid nanoparticle.
  • the functional human protein can be any one of proteins described in TABLE 11.
  • the functional human protein can be any one of proteins described in TABLE 11.
  • compositions described herein comprising compositions described herein.
  • cell that comprises the target nucleic acid modified by compositions described herein.
  • the systems comprise one or more components, wherein 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, wherein the effector protein comprises an amino acid sequence having at least 90% sequence identity to any one of amino acid sequences recited in TABLE 1A and TABLE IB; (ii) a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, and wherein the target sequence comprises a nucleotide sequence within the safe harbor; and (iii) the donor nucleic acid encoding a transgene that comprises a functional human protein that is expressed upon incorporation into the human safe harbor locus, wherein the safe harbor comprises at least 90% sequence identity to any one of nucle
  • the effector protein comprises one or more amino acid substitutions relative to any one of the amino acid sequences recited in TABLE 1A and TABLE IB.
  • the one or more amino acid substitutions independently comprise one or more conservative substitutions, one or more nonconservative 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 effector protein comprises an amino acid sequence with at least 95% sequence identity to any one of the amino acid sequences recited in TABLE 1A and TABLE IB.
  • the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of SEQ ID NO: 592, 600 and 602, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231.
  • the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 9.
  • the spacer sequence is at least 90% identical to any one of nucleotide sequences recited in TABLE 5, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, 13, 47, and 228-231.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, 228-230, wherein the effector protein comprises one or more amino acid substitutions independently selected from E109R, H208R, K184R, K38R, L182R, Q183R, S108R, S198R, T114R, or a combination thereof.
  • the systems comprising an effector protein or a nucleic acid encoding the effector protein, a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, and a donor nucleic acid, wherein the system comprises a viral vector.
  • any two components of the system are provided in different solutions or containers.
  • at least two components of the system are administered separately or simultaneously.
  • FIGs. 1A-1F shows indel activity of the effector protein CasPhi. 12 L26R (SEQ ID NO: 228) for gRNA within intron 1 of human albumin, as described in Example 1;
  • FIG. 7A-7B illustrates the effects of an arginine substitution on CasM.265466 nuclease activity for a target nucleic acid, in accordance with an embodiment of the present disclosure.
  • FIG. 16A shows indel activity of the effector protein system (e.g., CasPhi.12 L26R, CasM.265466) within or adjacent to intron 1 of human albumin gene as related to different concentrations of RNA and MOI and as compared to positive control (e.g., SpyCas9).
  • FIG. 16B shows relative light units (RLU) as a measure of integration activity of the effector protein system (e.g., CasPhi.12 L26R, CasM.265466) within or adjacent to intron 1 of human albumin gene as related to different concentrations of RNA and MOI and as compared to positive control (e.g., SpyCas9).
  • RLU relative light units
  • FIG. 17 shows % integration products as a measure of integration activity of the effector protein system (e.g., CasPhi.12 L26R, CasM.265466) within or adjacent to intron 1 of human albumin gene as compared to positive control (e.g., SpyCas9) as measured via reverse transcription droplet digital PCR (RT-ddPCR).
  • the effector protein system e.g., CasPhi.12 L26R, CasM.265466
  • positive control e.g., SpyCas9
  • RT-ddPCR reverse transcription droplet digital PCR
  • base editor refers to a fusion protein comprising a base editing enzyme fused to an effector protein.
  • 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.
  • the base editing enzyme may comprise deaminase activity. Additional base editors are described herein.
  • 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.
  • ssDNA or ssRNA single-stranded
  • dsDNA double-stranded
  • donor nucleotide refers to a single nucleotide that is incorporated into a target nucleic acid. A nucleotide is typically inserted at a site of cleavage by an effector protein.
  • fusion effector protein refers to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein. In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein.
  • fusion partner protein and “fusion partner,” as used herein, refer to a protein, polypeptide or peptide that is fused to an effector protein.
  • the fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein.
  • the fusion partner may modify a target nucleic acid, including changing a nucleobase of the target nucleic acid and making a chemical modification to one or more nucleotides of the target nucleic acid.
  • intermediary RNA and “intermediary sequence,” as used herein, 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.
  • linked amino acids refers to at least two amino acids linked by an amide bond.
  • linker refers to a bond or molecule that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid.
  • a “peptide linker” comprises at least two amino acids linked by an amide bond.
  • DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • sequences can be provided in the form of an open reading frame uninterrupted by internal non translated sequences, or introns, which are typically present in eukaryotic genes.
  • Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit.
  • 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 may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence.
  • the non-naturally occurring sequence is generated by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more engineered modifications, or both, relative to a naturally occurring or wildtype protein. 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.
  • M N-terminal Methionine
  • V Valine
  • compositions, systems, and methods comprising a polypeptide (e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof).
  • a polypeptide e.g., an effector protein, an effector partner, a fusion protein, or a combination thereof.
  • compositions, systems, and methods described herein comprise an effector protein or a use thereof.
  • the effector protein is a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that interacts with a target nucleic acid.
  • Modification activity of an effector protein or an engineered protein described herein may be cleavage activity, binding activity, insertion activity, substitution activity, and the like. Modification activity of an effector protein may result 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 proximity of 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 sequence in the target nucleic acid comprises introducing or removing epigenetic modification(s).
  • the modification of the target nucleic acid generated by an effector protein may, as a nonlimiting example, result 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).
  • methods of editing a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof are also provided herein are methods of modulating expression of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof.
  • methods of modulating the activity of a translation product of a target nucleic acid using an effector protein of the present disclosure, or compositions or systems thereof are provided herein.
  • effector proteins disclosed herein may provide nucleic acid cleavage activity, such as cis cleavage activity, trans cleavage activity, nickase activity, nuclease activity, or a combination thereof. Effector proteins disclosed herein may 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
  • An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary with a target sequence in the target nucleic acid.
  • the ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid.
  • An effector protein may also recognize (e.g., non-covalently interact) a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein.
  • PAM protospacer adjacent motif
  • an effector protein when functioning in a multiprotein complex, may have differing and/or complementary functional activity to other effector proteins in the multiprotein complex.
  • the functional activity includes but not limited to substrate selectivity, specificity, and/or affinity.
  • An effector protein may be a modified effector protein having reduced modification activity (e.g., a catalytically defective effector protein) or no modification activity (e.g. , a catalytically inactive effector protein).
  • an effector protein as used herein encompasses a modified or programmable nuclease (e.g., effector protein) that does not have nuclease activity.
  • the complementary functional activity of effector proteins comprising modified or artificial base pairs can be based on other types of hydrogen bonding and/or hydrophobicity of bases and/or shape complementarity between bases. Multimeric complexes, and functions thereof, are described in further detail below.
  • An effector protein may be 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 may be a catalytically inactive effector protein having reduced modification activity or no modification activity.
  • the amino acid sequence of the effector protein 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 recited in TABLE 1A and TABLE IB.
  • an effector protein provided herein comprises an amino acid sequence that is at least 65% identical to the amino acid sequence recited in TABLE 1A and TABLE IB.
  • an effector protein provided herein comprises an amino acid sequence that is at least 70% identical to the sequence recited in TABLE 1A and TABLE IB.
  • an effector protein provided herein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence recited in TABLE 1A and TABLE IB. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 98% identical to the amino acid sequence recited in TABLE 1A and TABLE IB. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is at least 99% identical to the amino acid sequence recited in TABLE 1A and TABLE IB. In some embodiments, an effector protein provided herein comprises an amino acid sequence that is identical to the amino acid sequence recited in TABLE 1A and TABLE IB
  • 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 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 1- 231.
  • the amino acid sequence of the effector protein 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 SEQ ID NO: 1- 231.
  • compositions, systems and methods described herein comprise an effector protein comprising an amino acid sequence for which 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 amino acid sequences and multiplying by 100.
  • percent identity e.g., % identity
  • the non-covalent interaction between macromolecules is referred to as binding or associating.
  • binding or associating are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions.
  • a nucleotide sequence that is able to noncovalently interact or anneal to another nucleotide sequence in a sequence-specific, antiparallel, manner is considered to be hybridizable or capable of hybridization.
  • the nucleotide sequence is able to noncovalently interact or anneal to another nucleotide sequence, as detailed herein, under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • 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.
  • an effector protein may be small, which may be beneficial for nucleic acid detection or editing (for example, the effector protein may be less likely to adsorb to a surface or another biological species due to its small size). The smaller nature of these effector proteins may allow for them to be more easily packaged and delivered with higher efficiency in the context of genome editing and more readily incorporated as a reagent in an assay.
  • the length of the effector protein is at least 400 linked amino acid residues. In some embodiments, the length of the effector protein is less than 500 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 500 linked amino acid residues.
  • the amino acid sequence of an effector protein provided herein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400 contiguous amino acids, at least about 420 contiguous amino acids, at least about 440 contiguous amino acids, at least about 460 contiguous amino acids, at least about 480 contiguous amino acids, at least about 500 contiguous amino acids, at least about 520 contiguous amino acids, at least about 540 contiguous amino acids, at least about 560 contiguous amino acids, at least about 580 contiguous amino acids, at least about 600 contiguous amino acids, at least about 620 contiguous amino acids, at least about 640 contiguous amino acids, at least about 660 contiguous amino acids, at least about 680 contiguous amino acids, at least about 700 contiguous amino acids, or more of the amino acids, at least about 420
  • an effector protein may be able to recognize a variety of PAMs as described herein.
  • effector proteins described herein may provide blunt or short stagger ends. Blunt cutting may be advantageous over the staggered cutting that is provided by other effector proteins, as there is a less likely chance of spontaneous (also referred to as perfect) repair which may decrease the chances of successful target nucleic acid editing and/or donor nucleic acid insertion.
  • 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), Vai (V), Leu (L), He (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), Vai (V), Leu (L), He (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), Gin (Q), Ser (S), Thr (T).
  • 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 1A and TABLE IB. In some embodiments, 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 1A and TABLE IB.
  • the variant comprises one or more amino acid alterations in a RuvC domain, a REC domain, TPID, NTPID, or combinations thereof.
  • the one or more alterations are one or more substitutions with Lys (K), Arg (R), or His (H).
  • variants comprise at least one alteration relative to any one of the amino acid sequences of TABLE 1A and TABLE IB, wherein the at least one alteration is a substitution with 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 amino acid sequence of the effector protein 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 any one of the amino acid sequences recited in TABLE 1A and TABLE IB.
  • 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: 229, wherein the effector protein has His (H) amino acid at position 26.
  • 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: 229, wherein has His (H) amino acid at position 26.
  • the effector protein comprises one or more amino acid substitutions independently selected from E109R, H208R, K184R, K38R, L182R, Q183R, S108R, S198R, T114R or a combination thereof, wherein the amino acid sequence of the effector protein 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 any one of SEQ ID NO: 6, and 228-230.
  • 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, wherein the amino acid sequence of the effector protein 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: 231.
  • the one or more amino acid alterations may result in a change in activity of the effector protein relative to a WT counterpart.
  • the one or more amino acid alteration increases or decreases catalytic activity of the effector protein relative to a WT counterpart.
  • the one or more amino acid alterations results in a catalytically inactive effector protein variant.
  • the effector proteins function as an endonuclease that catalyzes cleavage within a target nucleic acid.
  • the effector proteins are capable of catalyzing nonsequence -specific cleavage of a single stranded nucleic acid.
  • the effector proteins e.g., the effector proteins having the amino acid sequence of TABLE 1A and TABLE IB
  • the effector proteins are activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity may also be referred to as “collateral” or “transcollateral” cleavage.
  • Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated effector protein, such as trans cleavage of detector nucleic acids with a detection moiety.
  • Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide RNA (e.g., a dual guide nucleic acid system or a sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide RNA.
  • Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide RNA. Trans cleavage may occur near, but not within or directly adjacent to, the region of the target nucleic acid that is hybridized to the guide nucleic acid. Trans cleavage activity may be triggered by the hybridization of the guide nucleic acid to the target nucleic acid.
  • nickase activity is a selective cleavage of one strand of a dsDNA.
  • fusion proteins are targeted by a guide nucleic acid (guide RNA) to a specific location in the target nucleic acid.
  • a fusion partner comprises a subcellular localization signal.
  • a subcellular localization signal can be a nuclear localization signal (NLS).
  • 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. TABLE 2 lists exemplary NLS sequences.
  • the heterologous polypeptide 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 an effector 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.
  • compositions 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 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 1-231, wherein at least one of the N or C terminus comprises at least one of nuclear localization signal sequence recited in TABLE 2.
  • an endosomal escape protein comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 1823).
  • the amino acid sequence of the endosomal escape protein is GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 1822), wherein each X is independently selected from lysine, histidine, and arginine or GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 1823).
  • effector proteins described herein are encoded by a codon optimized nucleic acid.
  • compositions, systems, and methods described herein comprise a nucleic acid encoding an effector protein described herein, wherein the nucleic acid is codon optimized.
  • a nucleic acid encoding an effector protein described herein may be codon optimized for expression in a specific cell (e.g., a eukaryotic cell, an animal cell, a mammalian cell, or a human cell).
  • a nucleic acid encoding the effector protein described herein is codon optimized for a human cell.
  • the nucleic acid encoding the effector protein described herein comprises a mutation to mimic the codon preferences of the intended host organism or cell while encoding the same effector protein.
  • the codons can be changed, but the encoded effector protein remains unchanged.
  • 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, then a mouse codon-optimized nucleotide sequence encoding an effector protein could be generated.
  • 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.
  • An engineered protein may comprise a modified form of a wildtype counterpart protein (e.g., an effector protein).
  • the modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, substitution, or combination thereof) that reduces the nucleic acid-cleaving activity of the effector protein relative to the wildtype counterpart.
  • a nuclease domain e.g., RuvC domain
  • an effector protein may be deleted or mutated relative to a wildtype counterpart effector protein so that it is no longer functional or comprises reduced nuclease activity.
  • the modified form of the effector protein may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart.
  • Engineered proteins may have no substantial nucleic acid-cleaving activity.
  • Engineered proteins may be enzymatically inactive or “dead,” that is it may bind to a nucleic acid but not cleave it.
  • An enzymatically inactive protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain).
  • an effector protein that has decreased catalytic activity relative to a naturally occurring counterpart may be referred to as catalytically inactive, enzymatically inactive, catalytically dead, or enzymatically dead.
  • An effector protein that has decreased catalytic activity relative to a naturally occurring counterpart may be referred to as a dead protein or a dCas protein.
  • a dCas protein comprises an enzymatically inactive domain (e.g. inactive nuclease domain).
  • a nuclease domain e.g, RuvC domain
  • an effector protein may be deleted or mutated relative to a wildtype counterpart so that it is no longer functional or comprises reduced nuclease activity.
  • effector proteins disclosed herein comprise a truncated RuvC domain.
  • reduced nuclease activity may refer to an activity less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 100% activity compared to the wild-type counterpart.
  • a catalytically inactive effector protein may bind to a guide nucleic acid and/or a target nucleic acid but does not cleave the target nucleic acid.
  • a catalytically inactive effector protein may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid.
  • a catalytically inactive effector protein is fused to an effector partner that confers an alternative activity to an effector protein activity. Such fusion proteins are described herein and throughout.
  • the effector protein may comprise an enzymatically inactive and/or “dead” (abbreviated by “d”) effector protein in combination (e.g. , fusion) with a polypeptide comprising recombinase activity.
  • d enzymatically inactive and/or “dead”
  • nuclease-dead effector protein may also be referred to as a catalytically inactive effector protein.
  • an effector protein normally has nuclease activity, in some embodiments, an effector protein does not have nuclease activity.
  • an effector protein comprising a nuclease-dead effector protein, wherein the nuclease-dead effector protein comprising 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% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB.
  • the effector protein comprising 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% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB, wherein the effector protein is modified or engineered to be a nuclease- dead effector protein.
  • a nuclease-dead 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: 6, and wherein the effector protein further comprises one or more alterations selected from D369A, D369N, E567A, E567Q, D658A and D658N.
  • a nuclease- dead 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% identical to SEQ ID NO: 6, and wherein the effector protein further comprises E567A substitution.
  • a nuclease-dead 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% identical to SEQ ID NO: 6, and wherein the effector protein further comprises E567Q substitution.
  • a nuclease- dead 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% identical to SEQ ID NO: 6, and wherein the effector protein further comprises E567Q substitution.
  • a nuclease-dead 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% identical to SEQ ID NO: 7, and wherein the effector proteins comprise alanine amino acid at position 369.
  • a nuclease-dead 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: 7, and wherein the effector proteins comprise alanine amino acid at position 369.
  • a nuclease-dead 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: 8, and wherein the effector protein comprises asparagine amino acid at position 369.
  • a nuclease-dead 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% identical to SEQ ID NO: 10, and wherein the effector protein comprises glutamine amino acid at position 567.
  • a nuclease-dead 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: 10, and wherein the effector protein comprises glutamine amino acid at position 567.
  • a nuclease-dead 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% identical to SEQ ID NO: 231, and wherein the effector protein further comprises one or more alterations selected from D237A, D418A, D418N, E335A, and E335Q.
  • compositions, systems, and methods comprise a fusion protein or uses thereof.
  • a fusion protein generally comprises an effector protein and a fusion partner protein.
  • a fusion partner protein is also simply referred to herein as a fusion partner.
  • the fusion partner is a heterologous protein capable of imparting some function or activity that is not provided by the effector protein.
  • the fusion partner comprises a polypeptide or peptide that is fused or linked to the effector protein.
  • the fusion partner is fused or linked to the effector protein.
  • the fusion partner is fused to the N-terminus of the effector protein.
  • the fusion partner is fused to the C-terminus of the effector protein.
  • the fusion protein is a heterologous peptide or polypeptide as described herein.
  • the amino terminus of the fusion partner is linked/fused to the carboxy terminus of the effector protein.
  • the carboxy terminus of the fusion partner protein is linked/fused to the amino terminus of the effector protein by the linker.
  • the fusion partner is not an effector protein as described herein.
  • the fusion partner comprises an effector protein as described herein or a multimeric form thereof. Accordingly, in some embodiments, the fusion protein comprises more than one effector protein.
  • a fusion partner may inhibit or promote the formation of multimeric complex of an effector protein.
  • the fusion partner may directly or indirectly edit a target nucleic acid. Modifications can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid.
  • the fusion partner may interact with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid.
  • Modifications can be of a nucleobase, nucleotide, or nucleotide sequence of a target nucleic acid.
  • the fusion partner may interact with additional proteins, or functional fragments thereof, to make modifications to a target nucleic acid.
  • 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 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 may direct the modification activity of a fusion protein.
  • Modification activity of a fusion protein described herein may be cleavage activity, binding activity, insertion activity, substitution activity, and the like. Modification activity of an effector protein may result 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.
  • fusion partners provide enzymatic activity that modifies a target nucleic acid.
  • enzymatic activities include, but are not limited to, nuclease activity, demethylase activity, DNA repair activity, deamination activity, dismutase activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.
  • fusion partners have enzymatic activity that modifies the target nucleic acid.
  • the target nucleic acid may comprise or consist of a ssRNA, dsRNA, ssDNA, or a dsDNA.
  • transposase activity refers to catalytic activity that results in the transposition of a first nucleic acid into a second nucleic acid.
  • Non-limiting examples of fusion partners for targeting ssRNA include, but are not limited to, protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); helicases; and RNA-binding proteins. It is understood that a fusion protein may include the entire protein or in some embodiments may include a fragment of the protein (e.g. , a functional domain). In some embodiments, the functional domain interacts with or binds ssRNA, including intramolecular and/or intermolecular secondary structures thereof, e.g., hairpins, stem-loops, etc.). The functional domain may interact transiently or irreversibly, directly or indirectly.
  • protein translation components e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G
  • helicases e.g., helicases
  • RNA-binding proteins e.g., RNA-binding
  • the effector domain may be a domain of a protein selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; and proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity.
  • fusion partners comprise a recombinase.
  • effector proteins described herein are fused with the recombinase.
  • the effector proteins have reduced nuclease activity or no nuclease activity.
  • the recombinase is a sitespecific recombinase.
  • 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.
  • a linker comprises a bond or molecule that links a first polypeptide to a second polypeptide.
  • effector proteins, fusion proteins, fusion partners, or combinations thereof are connected by linkers.
  • effector proteins and fusion partners of a fusion effector protein are connected by a linker.
  • the linker may comprise or consist of a covalent bond.
  • the linker may comprise or consist of a chemical group.
  • the linker comprises an amino acid.
  • 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 fusion partner.
  • linkers comprise one or more amino acids.
  • a linker comprises a peptide.
  • a terminus of the effector protein is linked to a terminus of the fusion partner through an amide bond.
  • a terminus of the effector protein is linked to a terminus of the fusion partner through a peptide bond.
  • linkers comprise an amino acid.
  • linkers comprise one or more amino acids.
  • linkers comprise a peptide.
  • linker is a protein.
  • an effector protein is coupled to a fusion partner via a linker protein.
  • the XTEN linker is an XTEN80 linker. In some embodiments, the XTEN linker is an XTEN20 linker. In some embodiments, the XTEN20 linker has an amino acid sequence of GSGGSPAGSPTSTEEGTSESATPGSG (SEQ ID NO: 1790).
  • linkers do not comprise an amino acid. In some embodiments, linkers do not comprise a peptide. In some embodiments, linkers comprise a nucleotide, a polynucleotide, a polymer, or a lipid.
  • a linker is recognized and cleaved by a protein.
  • a linker comprises a recognition sequence that may be recognized and cleaved by the protein.
  • a guide nucleic acid comprises an aptamer, which may serve a similar function as a linker, bringing an effector protein and an effector partner protein into proximity.
  • the aptamer can functionally connect two proteins (e.g., effector protein, effector partner) 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 capable of being bound be the aptamer binding moiety.
  • the aptamer is a molecule that is capable of mimicking antibody binding activity and may be 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 pM range) with little or no off-target binding.
  • compositions, systems, and methods of the present disclosure may comprise a multimeric complex or uses thereof, wherein the multimeric complex comprises multiple effector proteins that non- covalently interact with one another.
  • a multimeric complex may comprise enhanced activity relative to the activity of any one of its effector proteins alone.
  • a multimeric complex comprising two effector proteins may comprise greater nucleic acid binding affinity, cis-cleavage activity, and/or transcollateral cleavage activity than that of either of the effector proteins provided in monomeric form.
  • a multimeric complex may have an affinity for a target region of a target nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking, editing or modifying the nucleic acid) at or near the target region.
  • Multimeric complexes may be activated when complexed with a guide nucleic acid. Multimeric complexes may be activated when complexed with a guide nucleic acid and a target nucleic acid. In some embodiments, the multimeric complex cleaves the target nucleic acid. In some embodiments, the multimeric complex nicks the target nucleic acid.
  • multimeric complexes comprise at least one effector protein, or a fusion protein thereof, comprising 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 recited in TABLE 1A and TABLE IB.
  • multimeric complexes comprise at least one effector protein or a fusion protein thereof, wherein the amino acid sequence of the effector protein 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 recited in TABLE 1A and TABLE IB
  • the multimeric complex is a dimer comprising two effector proteins of identical amino acid sequences.
  • the multimeric complex comprises a first effector protein and a second effector protein, wherein the amino acid sequence of the first effector protein is at least 90%, at least 92%, at least 94%, at least 96%, at least 98% identical, or at least 99% identical to the amino acid sequence of the second effector protein.
  • the multimeric complex is a heterodimeric complex comprising at least two effector proteins of different amino acid sequences.
  • 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.
  • a multimeric complex comprises at least two effector proteins. In some embodiments, a multimeric complex comprises more than two effector proteins. In some embodiments, a multimeric complex comprises two, three or four effector proteins. In some embodiments, at least one effector protein of the multimeric complex 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 recited in TABLE 1A and TABLE IB.
  • each effector protein of the multimeric complex 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 recited in TABLE 1A and TABLE IB.
  • Any suitable method of generating and assaying effector proteins described herein may be used. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2nd ed (2014)).
  • compositions, systems, and methods described herein may further comprise a purification tag that can be attached to an effector protein, or a nucleic acid encoding the purification tag that can be attached to a nucleic acid encoding the effector protein as described herein.
  • the purification tag may be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which may be its biological source, such as a cell lysate. Attachment of the purification tag may be at the N or C terminus of the effector protein.
  • effector proteins described herein are isolated from cell lysate.
  • the compositions described herein may comprise 20% or more by weight, 75% or more by weight, 95% or more by weight, or 99.5% or more by weight of an effector protein, related to the method of preparation of compositions described herein and its purification thereof, wherein percentages may be upon total protein content in relation to contaminants.
  • the effector protein is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-engineered polypeptide proteins or other macromolecules, etc.).
  • Effector proteins of the present disclosure may cleave or nick 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.
  • effector proteins 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 effector protein does not require a PAM to bind and/or cleave a 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.
  • the PAM sequence is located on the non-target strand.
  • 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, such a PAM described herein is directly adjacent to the target sequence on the target strand or the non-target strand.
  • an effector protein described herein, or a multimeric complex thereof recognizes a PAM on a target nucleic acid.
  • multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid.
  • at least two of the multiple effector proteins recognize the same PAM sequence.
  • at least two of the multiple effector proteins recognize different PAM sequences.
  • only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid.
  • Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid.
  • PAM protospacer adjacent motif
  • cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides of a 5’ or 3’ terminus of a PAM sequence.
  • a target nucleic acid may comprise a PAM sequence adjacent to a nucleotide sequence that is complementary to a guide nucleic acid spacer region.
  • the effector protein recognizes a PAM sequence.
  • the PAM sequence comprises at least three nucleotides.
  • the PAM sequence comprises at least four nucleotides. In some embodiments, the PAM sequence comprises at least two consecutive thymine bases. In some embodiments, the PAM sequence comprises at least three consecutive thymine bases. Each, nucleotide and nucleoside, describes sugar and base of the residue contained in the nucleic acid molecule.
  • the PAM sequence comprises any of the nucleotide sequences recited in TABLE 3.
  • a composition comprising an effector protein recognizes a PAM sequence comprising any of the nucleotide sequences recited in TABLE 3.
  • effector proteins described herein recognize any one of PAM sequences recited in TABLE 3. In some embodiments, effector proteins described herein recognize any one of PAM sequences of SEQ ID NO: 234-251. In some embodiments, the PAM sequence is within 20, 18, 16, 14, 12, 10, 8, 6, 4 or 2 bases ofthe target sequence. In some embodiments, compositions and systems described herein comprises an effector protein having 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 any one of SEQ ID NO: 1-231, and that recognizes any one of PAM sequences of SEQ ID NO: 234-251.
  • compositions and systems described herein comprises an effector protein having 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: 13, and that recognizes any one of PAM sequences of SEQ ID NO: 238-247.
  • compositions and systems described herein comprises an effector protein having 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: 47, and that recognizes any one of PAM sequences of SEQ ID NO: 241-244.
  • compositions and systems described herein comprises an effector protein having 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: 187 or 188, and that recognizes a PAM sequence of SEQ ID NO: 250.
  • nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid.
  • disclosure of the nucleotide sequences described herein also discloses a complementary nucleotide sequence, a 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.
  • a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein.
  • Guide nucleic acids can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone.
  • Guide nucleic acids may be referred to herein as a guide RNA (gRNA).
  • gRNA guide RNA
  • a guide RNA is not limited to ribonucleotides, but may comprise deoxyribonucleotides and other chemically modified nucleotides.
  • compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid, a nucleic acid encoding the guide nucleic acid, or a use thereof.
  • 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.
  • Guide nucleic acids are also referred to herein as “guide RNA.”
  • a guide nucleic acid, as well as any components thereof may comprise one or more deoxyribonucleotides (DNA), ribonucleotides (RNA), a combination thereof (e.g., RNA with a thymine base), 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 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 sequence that encodes a guide nucleic acid, such as a nucleotide sequence described herein for a vector.
  • uridine for thymidine or vice versa
  • nucleoside analogs such as modified uridines
  • adenosine for all of thymidine, uridine, or modified uridine
  • cytosine and 5- methylcytosine both of which have guanosine or modified guanosine as a complement
  • a guide nucleic acid may comprise a naturally occurring guide nucleic acid.
  • a guide nucleic acid may comprise a non-naturally occurring guide nucleic acid, including a guide nucleic acid that is designed to contain a chemical or biochemical modification.
  • 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.
  • the guide RNA may be chemically synthesized or recombinantly produced.
  • the sequence of the guide nucleic acid, or a portion thereof may be different from the sequence of a naturally occurring nucleic acid.
  • Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism.
  • a portion of the complex when in a complex, at least a portion of the complex may bind, recognize, and/or hybridize 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 may hybridize 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 may comprise or form intramolecular secondary structure (e.g., hairpins, stem-loops, etc.).
  • aguide nucleic acid comprises a stemloop 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).
  • a nucleic acid having multiple linked nucleotides or nucleosides describes linked sugars and bases of residues contained in the nucleic acid molecule.
  • An effector protein may recognize 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.
  • 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.
  • the compositions, systems, and methods of the present disclosure may comprise an additional guide nucleic acid or a use thereof.
  • An additional guide nucleic acid can target an effector protein to a different location in the target nucleic acid by binding to a different portion of the target nucleic acid from the first guide nucleic acid.
  • a guide nucleic acid can bind a portion of the target nucleic acid that is upstream or downstream of the target gene in a cell or subject as described herein, wherein the additional guide nucleic acid can bind to a portion of the target nucleic acid that is located either upstream or downstream of where the first guide RNA has targeted.
  • the first loci and the second loci of the target nucleic acid may be 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 may be 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.
  • the first loci and/or the second loci of the target nucleic acid are located in an intron of a gene.
  • the first loci and/or the second loci of the target nucleic acid are located in an exon of a gene .
  • the first loci and/or the second loci of the target nucleic acid span an exonintronjunction of a gene.
  • compositions, systems, and methods comprise a donor nucleic acid that may be 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 may be identical, nonidentical, or combinations thereof.
  • 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. In some embodiments, 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.
  • guide nucleic acids comprise additional elements that contribute additional functionality (e.g., stability, heat resistance, etc.) to the guide nucleic acid.
  • Such elements may be 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 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides.
  • a linker may be any suitable linker, examples of which are described herein.
  • the guide nucleic acid comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked nucleosides.
  • a guide nucleic acid comprises at least 10 linked nucleosides.
  • a guide nucleic acid comprises at least 25 linked nucleosides.
  • a guide nucleic acid may comprise 10 to 50 linked nucleosides.
  • the guide nucleic acid comprises or consists essentially of about 12 to about 80 linked nucleosides, about 12 to about 50, about 12 to about 45, about 12 to about 40, about 12 to about 35, about 12 to about 30, about 12 to about 25, from about 12 to about 20, about 12 to about 19 , about 19 to about 20, about 19 to about 25, about 19 to about 30, about 19 to about 35, about 19 to about 40, about 19 to about 45, about 19 to about 50, about 19 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, or about 20 to about 60 linked nucleosides.
  • the guide nucleic acid has about 10 to about 60, about 20 to about 50, or about 30 to about 40 linked nucleosides.
  • nucleotide sequences described herein may be described as a nucleotide sequence of either DNA or RNA, however, no matter the form the 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 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.
  • the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a sequence that is at least 70%, 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 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, TABLE 9 or any combination thereof.
  • the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NO: 1825, 1828, 1846 and 1847.
  • the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NO: 652, 685, 705, 713, 777 and 790. In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NO: 856, 864 and 866.
  • the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a spacer sequence and/or a repeat sequence. In some embodiments, the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a spacer sequence and/or a handle sequence. In some embodiments, the handle sequence comprises one or more of an intermediary sequence, a tracrRNA sequence or a portion thereof, a repeat sequence, and a linker.
  • the nucleotide sequence of the guide nucleic acid 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 nucleotide sequences recited in TABLE 13, TABLE 14, TABLE 15, TABLE 16, TABLE 17, TABLE 19, TABLE 20, and TABLE 21.
  • an exemplary composition may comprise an effector protein comprising an amino acid sequence of SEQ ID NO: 6 or 228 recognizing any one of PAM sequences recited in TABLE 13, and a guide nucleic acid 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 nucleotide sequences recited in TABLE 13.
  • an exemplary composition may comprise an effector protein comprising an amino acid sequence of SEQ ID NO: 228 recognizing any one of PAM sequences recited in TABLE
  • a guide nucleic acid 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 nucleotide sequences recited in TABLE 16.
  • an exemplary composition may comprise an effector protein comprising any one of amino acid sequences of SEQ ID NO: 6 and 228- 230 recognizing any one of PAM sequences recited in TABLE 17, and a guide nucleic acid comprising a repeat sequence and a spacer sequence, wherein the repeat sequence 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 SEQ ID NO: 252 and the spacer sequence 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 spacer sequences recited in TABLE 17.
  • an exemplary composition may comprise an effector protein comprising an amino acid sequence of SEQ ID NO: 13 recognizing any one of PAM sequences recited in TABLE 19, and a guide nucleic acid comprising a handle sequence and a spacer sequence, wherein the handle sequence 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 handle sequences recited in TABLE 19 and the spacer sequence 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 spacer sequences recited in TABLE 19.
  • an exemplary composition may comprise an effector protein comprising an amino acid sequence of SEQ ID NO: 47 recognizing any one of PAM sequences recited in TABLE 20, and a guide nucleic acid comprising a repeat sequence and a spacer sequence, wherein the repeat sequence 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 SEQ ID NO: 254 and the spacer sequence 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 spacer sequences recited in TABLE 20.
  • an exemplary composition may comprise an effector protein comprising any one of amino acid sequences of SEQ ID NO: 181-184 recognizing any one of PAM sequences recited in TABLE 21, and a guide nucleic acid comprising a handle sequence and a spacer sequence, wherein the handle sequence 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 handle sequences recited in TABLE 21 and the spacer sequence 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 spacer sequences recited in TABLE 21.
  • a guide nucleic acid comprises a spacer region that hybridizes to a target sequence of a target nucleic acid.
  • the spacer region may comprise complementarity with (e.g., hybridize to) a target sequence of a target nucleic acid.
  • 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 at least about 25, or at least about 15 to about 25 linked nucleotides.
  • the spacer region is 15-28 linked nucleosides in length. In some embodiments, the spacer region is 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 nucleosides in length. In some embodiments, the spacer region is 18- 24 linked nucleosides in length. In some embodiments, the spacer region is at least 15 linked nucleosides in length. In some embodiments, the spacer region is at least 16, 18, 20, or 22 linked nucleosides in length.
  • the spacer region 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 nucleotides. In some embodiments, the spacer region is at least 17 linked nucleosides in length. In some embodiments, the spacer region is at least 18 linked nucleosides in length. In some embodiments, the spacer region is at least 20 linked nucleosides in length. In some embodiments, the spacer region is at least 80 %, at least 85 %, at least 90 %, at least 95 % or 100 % complementary to a target sequence of the target nucleic acid.
  • Partially complementarity nucleotide sequence comprises at least 20%, but less than 100%, of the residues of the nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some embodiments, at least 50%, but less than 100%, of the residues of a nucleotide sequence are complementary to residues in a reference nucleotide sequence. In some embodiments, 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 nucleotide sequences comprises less than 20% of the residues of the nucleotide sequence are complementary to residues in a reference nucleotide sequence.
  • the spacer region is 100 % complementary to the target sequence of the target nucleic acid. In some embodiments, the spacer region comprises at least 15 contiguous nucleotides that are complementary to the target nucleic acid. In some embodiments, the spacer region comprises at least 17 contiguous nucleotides that are complementary to the target nucleic acid.
  • a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, 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. Linkers may be 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.
  • nucleotide sequence of a spacer region 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 guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 20 of the spacer region that is not complementary to the corresponding nucleoside of the target sequence.
  • the guide nucleic acid may comprise at least one uracil between nucleic acid residues 5 to 9, 10 to 14, or 15 to 20 of the spacer region that is not complementary to the corresponding nucleoside of the target sequence.
  • 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 is capable of hybridizing to an equal length portion of a target nucleic acid (e.g., a target sequence).
  • the region of the target nucleic acid that is complementary to the spacer region comprises an epigenetic modification or a post-transcriptional modification.
  • the epigenetic modification comprises an acetylation, methylation, or thiol modification.
  • a target nucleic acid comprises any one of the nucleotide sequences recited in TABLE 10.
  • a spacer sequence 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 atarget sequence of any one of target nucleic acids recited in TABLE 10.
  • the spacer sequence may comprise at least one modification, such as substituted or modified nucleotide, that is not complementary to the corresponding nucleotide of the target sequence.
  • 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 capable of hybridizing 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. In some embodiments, a spacer region is capable of hybridizing to a target sequence within a target nucleic acid, wherein the target nucleic acid is a safe harbor. In some embodiments, the spacer region is capable of hybridizing to a target sequence within a safe harbor loci.
  • the safe harbor locus can be any one of the loci of AAVS1 (PPP1R12C), ALB, Angptl , ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpinal, TF, or TTR.
  • the guide nucleic acid comprises 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% or 100% identical to any one of the nucleotide sequences recited in TABLE 4 and TABLE 5.
  • the spacer 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 any one of the nucleotide sequences recited in TABLE 4, wherein the spacer sequence hybridizes to a target 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: 266.
  • the spacer sequence comprises an RNA 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 SEQ ID NO: 301-604. In some embodiments, the spacer sequence comprises an RNA 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 SEQ ID NO: 348, 558, 401, 409, 473 and 486.
  • the spacer sequence comprises one or more nucleobase alterations at one or more positions in any one of the nucleotide sequences recited in TABLE 4.
  • Alternative nucleobases can be any one or more of A, C, G, T or U, or a deletion, or an insertion.
  • a target nucleic acid is a safe harbor within human SERPINA1 gene (SEQ ID NO: 1795).
  • the safe harbor comprises exon 1 of human SERPINA1 gene, intron 1 of human SERPINA1 gene, exon 2 of human SERPINA1 gene, or a combination thereof.
  • Guide nucleic acids described herein may 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 may interact with an effector protein.
  • a repeat sequence is connected to another sequence of a guide nucleic acid, such as an intermediary sequence, that is capable of non-covalently interacting with an effector protein.
  • a repeat sequence includes a nucleotide sequence that is capable of forming 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.
  • the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex).
  • dsRNA duplex double stranded RNA 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).
  • bp base pairs
  • not all nucleotides of the dsRNA duplex are paired, and, therefore, the duplex forming sequence may include a bulge.
  • the repeat sequence comprises a hairpin or stemloop 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.
  • such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity).
  • a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events may hybridize 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.).
  • a guide nucleic acid comprises a repeat region that interacts with the effector protein.
  • the repeat region may also be referred to as a “protein-binding segment.”
  • the repeat region is adjacent to the spacer region.
  • a guide RNA that interacts with an effector protein comprises a repeat region that is 5’ of the spacer region.
  • TABLE 6 provides illustrative repeat sequences for use with the compositions and methods of the disclosure.
  • the repeat sequence comprises at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of the nucleotide sequences as set forth in TABLE 6.
  • the repeat sequence comprises one or more nucleobase alterations at one or more positions in any one of the nucleotide sequences of TABLE 6.
  • Alternative nucleobases 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 crRNA sequence that is at least 65%, 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 SEQ ID NO: 252-258, 1789 and 1848.
  • compositions, systems and methods described herein comprise a crRNA comprising 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 252-258, 1789 and 1848.
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 6-12, and 228- 230; a crRNA comprising a repeat 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: 252 or 1848.
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 13; a crRNA comprising a repeat 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: 253
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 47; a crRNA comprising a repeat 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: 254
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 231; a crRNA comprising a repeat 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: 255 or 1789
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 181-184; a crRNA comprising a repeat 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: 256.
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 181-185; a crRNA comprising a repeat 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: 258.
  • Guide nucleic acids described herein may comprise one or more intermediary sequences.
  • an intermediary sequence used in the present disclosure is not transactivated or transactivating.
  • An intermediary sequence may also be referred to as an intermediary RNA, although it may comprise 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, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the intermediary sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.
  • An effector protein may interact 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 any one of the intermediary sequences in TABLE 7.
  • 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, 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, or at least 140 contiguous nucleotides of any one of the intermediary sequences recited in TABLE 7.
  • a sgRNA may include a handle sequence having a hairpin region, as well as a linker and a repeat sequence.
  • the sgRNA having a handle sequence can have a hairpin region positioned 3’ of the linker and/or repeat sequence.
  • the sgRNA having a handle sequence can have a hairpin region positioned 5’ of the linker and/or repeat sequence.
  • the hairpin region may include a first nucleotide sequence, a second nucleotide sequence that is reverse complementary to the first nucleotide sequence, and a stem -loop linking the first nucleotide sequence and the second nucleotide sequence.
  • the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length.
  • the sgRNA 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 may recognize a sgRNA comprising multiple stem regions.
  • the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the sgRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • a handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a length of the handle sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides.
  • the length of a handle sequence in a sgRNA is not greater than 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides.
  • the length of a handle sequence in a sgRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 66 to 105 linked nucleotides, 67 to 105 linked nucleotides, 68 to 105 linked nucleotides, 69 to 105 linked nucleotides, 70 to 105 linked nucleotides, 71 to 105 linked nucleotides, 72 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 40 to 70 nucleotides.
  • the length of a handle sequence in a sgRNA is 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in a sgRNA is 69 nucleotides.
  • TABLE 7 provides illustrative handle sequence for an sgRNA and exemplary portions of a sgRNA (a handle sequence without a linker or repeat sequence) for use with the compositions and methods of the disclosure.
  • the handle sequence comprises a sequence that is at least 65%, 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 nucleotide sequences recited in TABLE 7.
  • the handle sequence comprises at least 5, 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, or at least 140 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 7.
  • 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 same. In some embodiments, at least two of the more than one linker are not 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 of 5’- GAAA-3’.
  • a guide nucleic acid comprises one or more linkers connecting one or more of a repeat sequence, a spacer sequence, a handle sequence, and an intermediary sequence.
  • the guide nucleic acid comprises one or more linkers connecting one or more of: a repeat sequence and a spacer sequence; a handle sequence and a spacer sequence; an intermediary sequence and a repeat sequence; and an intermediary sequence and a spacer sequence.
  • the guide nucleic acid comprises at least two repeat sequences connected by a linker.
  • 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.
  • 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.
  • the guide 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 9, wherein an effector protein has 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 SEQ ID NO: 6-12, 228- 231, 47, and 13.
  • the guide 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 SEQ ID NO: 605-814, wherein an effector protein has 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 SEQ ID NO: 6-12, and 228-230.
  • the guide 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 SEQ ID NO: 815-839, wherein an effector protein has 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 SEQ ID NO: 47.
  • the guide 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 SEQ ID NO: 840-868, wherein an effector protein has 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 SEQ ID NO: 231.
  • a guide nucleic acid may comprise a CRISPR RNA (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.
  • the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, 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 may be the product of processing of a longer precursor CRISPR RNA (pre-crRNA) transcribed from the CRISPR array by cleavage of the pre-crRNA within each direct repeat sequence to afford shorter, mature crRNAs.
  • a crRNA may be generated by a variety of mechanisms, including the use of dedicated endonucleases (e.g., Cas6 or Cas5d in Type I and III systems), coupling of a host endonuclease (e.g., RNase III) with tracrRNA (Type II systems), or a ribonuclease activity endogenous to the effector protein itself (e.g., Cpfl, from Type V systems).
  • a crRNA may also be specifically generated outside of processing of a pre-crRNA and individually contacted to an effector protein in vivo or in vitro.
  • Exemplary crRNA sequences are recited in TABLE 9.
  • a crRNA or a nucleotide encoding the crRNA comprises a nucleotide sequence that is at least 65%, 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 crRNA sequences recited in TABLE 9.
  • a crRNA or a nucleotide encoding the crRNA comprises a nucleotide sequence that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides of any one of the crRNA sequences recited in TABLE 9.
  • the guide nucleic acid is a crRNA, wherein the crRNA has 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 SEQ ID NO: 605-839, wherein an effector protein has 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 SEQ ID NO: 6- 12, SEQ ID NO: 228-230, and SEQ ID NO: 47 sgRNA
  • compositions comprising a guide RNA and an effector protein without a tracrRNA (e.g. , a single nucleic acid system), wherein the guide RNA is a sgRNA.
  • a sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region).
  • a nucleotide sequence can be contained within a handle sequence as described herein.
  • a sgRNA comprises one or more of 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 an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and crRNA.
  • an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a crRNA are linked in an 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 an sgRNA.
  • a sgRNA comprises a linked handle sequence and spacer sequence.
  • a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g. , covalently linked, such as through a phosphodiester bond)
  • a handle sequence and a spacer sequence are linked in an 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 an sgRNA.
  • a sgRNA comprises a linked intermediary sequence and repeat sequence.
  • an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g. , covalently linked, such as through a phosphodiester bond)
  • an intermediary sequence and a repeat sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • a repeat sequence is 5’ to a spacer sequence in an sgRNA.
  • a sgRNA comprises a linked repeat sequence and spacer sequence.
  • a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond)
  • a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.
  • An exemplary handle sequence in a sgRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region.
  • the 5’ region may hybridize to the 3’ region.
  • the 5’ region does not hybridize to the 3’ region.
  • the 3’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).
  • the 5’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).
  • the sgRNA sequence comprises a nucleotide sequence that is at least 65%, 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 7.
  • compositions, systems and methods described herein comprise a handle sequence, wherein the handle sequence further comprises a linker.
  • the linker comprises any one of the nucleotide sequence recited in TABLE 8.
  • the linker comprises a sequence of SEQ ID NO: 265.
  • compositions, systems and methods described herein comprise sgRNA, wherein the sgRNA comprises a spacer sequence.
  • compositions, methods and systems described herein comprise a repeat sequence.
  • the repeat sequence comprises an RNA 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 252-258, 1789 and 1848.
  • compositions disclosed herein comprises 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%, at least 99%, or 100% identical to SEQ ID NO: 13; a guide nucleic acid comprising 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%, or 100% identical to any one of SEQ ID NO: 259-261.
  • compositions disclosed herein comprises 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%, at least 99%, or 100% identical to SEQ ID NO: 231; a guide nucleic acid comprising 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%, or 100% identical to any one of SEQ ID NO: 262-264.
  • the guide nucleic acid is a single guide RNA (sgRNA). Accordingly, in some embodiments, guide nucleic acids comprise a portion or all of any one of nucleotide sequences of SEQ ID NO: 252-264, or 1789, and 1848.
  • the guide nucleic acid is a sgRNA, wherein the sgRNA has 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 SEQ ID NO: 840-908, wherein an effector protein has 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 SEQ ID NO: 231, and 13.
  • a transactivating or transactivated nucleic acid comprises a first sequence that is capable of being non- covalently bound by an effector protein and a second sequence that is capable of hybridizing, at least partially, to a portion of a crRNA to form a tracrRNA-crRNA duplex in a dual nucleic acid system.
  • the second sequence that hybridizes to a portion of a crRNA may be referred to as a repeat hybridization sequence.
  • a transactivating or transactivated nucleic acid in a dual nucleic acid system is capable of hybridizing, at least partially, to a crRNA to form a tracrRNA-crRNA duplex, and of interacting with an effector protein to form a complex (e.g., an RNP complex).
  • a repeat hybridization sequence is a nucleotide sequence of a tracrRNA that is capable of hybridizing to a repeat sequence of a guide nucleic acid in the dual nucleic acid system.
  • the compositions comprising a guide RNA and an effector protein comprises a tracrRNA.
  • a tracrRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof.
  • a tracrRNA may be separate from, but form a complex with, a guide nucleic acid and an effector protein.
  • a tracrRNA may include a nucleotide sequence that hybridizes with a portion of a guide nucleic acid (e.g., a repeat hybridization region).
  • tracrRNAs 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 tracrRNA 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 may recognize a tracrRNA 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 tracrRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.
  • the length of a tracrRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 68 to 105 linked nucleotides, 71 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a tracrRNA is 40 to 60 nucleotides. In some embodiments, the length of a tracrRNA is 50, 56, 68, 71, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a tracrRNA is 50 nucleotides.
  • An exemplary tracrRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, a repeat hybridization region, and a 3’ region.
  • the 5’ region may hybridize to the 3’ region.
  • the 5’ region does not hybridize to the 3’ region.
  • a tracrRNA may comprise an un -hybridized region at the 3 ’ end of the tracrRNA.
  • the un-hybridized region may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides.
  • the length of the un-hybridized region is 0 to 20 linked nucleotides.
  • the repeat region may also be referred to as a “protein-binding segment.”
  • the repeat region is adjacent to the spacer region.
  • a guide RNA that interacts with an effector protein comprises a repeat region that is 5’ of the spacer region.
  • Polypeptides e.g., effector proteins
  • nucleic acids e.g., engineered guide nucleic acids
  • Polypeptides and nucleic acids can be further modified as described herein. Examples are modifications that do not alter the primary sequence of the polypeptides or nucleic acids, including chemical derivatization of polypeptides (e.g., acylation, acetylation, carboxylation, amidation, etc.).
  • 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. D-amino acids may be 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.
  • Modifications can further include the introduction of various groups to polypeptides and/or guide nucleic acids described herein.
  • 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.
  • cysteines may be 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.
  • 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: 2’0-methyl modified nucleotides, 2’ fluoro modified nucleotides; 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 phosphonates including 3'-alkylene phosphonates, 5 '-alkylene phosphonates and chiral phosphonates, phosphinates, phospho
  • an 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).
  • 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.
  • 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.
  • compositions, systems, and methods described herein comprise a vector or a use thereof.
  • a vector can comprise one or more nucleic acids.
  • 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 effector protein(s), fusion effector protein(s), fusion partner protein(s), guide nucleic acid(s), target nucleic acid(s), and donor nucleic acid(s).
  • 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 effector proteins.
  • 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,
  • a fusion effector protein as described herein is inserted into a vector.
  • a vector may comprise or encode one or more regulatory elements. Regulatory elements may refer 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
  • 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.
  • plasmids and vectors described herein comprise at least one promoter.
  • the promotors drive 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.
  • the promoters are constitutive promoters.
  • the promoters are inducible promoters.
  • 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).
  • promoters include, but are not limited to, CMV, 7SK, EFla, RPBSA, hPGK, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, Hl, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, MSCV, MNDU3, and HSV TK promoter.
  • the promoter is CMV.
  • the promoter is EFla.
  • the promoter is ubiquitin.
  • vectors are bicistronic or polycistronic vector (e.g.
  • the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, 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.
  • IPTG Isopropyl-beta-D-thiogalactopyranoside
  • the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44.
  • the promoter for expressing effector protein is a liver-specific promoter.
  • the liver-specific promoter comprises ApoE or TBG promoter sequence.
  • the promoter for expressing effector protein is a muscle-specific promoter.
  • the muscle-specific promoter comprises Ck8e, SPC5- 12, or Desmin promoter sequence.
  • the promoter for expressing effector protein is a ubiquitous promoter.
  • the ubiquitous promoter comprises MND or CAG promoter sequence.
  • a vector described herein is a nucleic acid expression vector. In some embodiments, a vector described herein is a recombinant expression vector. In some embodiments, a vector described herein is a messenger RNA.
  • the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g. , a bacterial vector) a viral vector, or any combination thereof.
  • the delivery vehicle may be a non-viral vector.
  • the delivery vehicle may be a plasmid.
  • the plasmid comprises DNA.
  • the plasmid comprises RNA.
  • the plasmid comprises circular double-stranded DNA.
  • the plasmid may be linear.
  • the plasmid comprises one or more genes 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 may be 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 plasmid may be formulated for delivery through injection by a needle carrying syringe.
  • the plasmid may be formulated for delivery via electroporation.
  • the plasmids may be engineered through synthetic or other suitable means known in the art.
  • the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.
  • the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic cell.
  • exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery.
  • Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; 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 effector proteins, 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 an effector protein are encoded by the same vector.
  • an effector protein (or a nucleic acid encoding same) and/or an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle.
  • an effector protein (or a nucleic acid encoding same), an engineered guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and/or 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 may be part of a vector system.
  • the vector system comprises a library of vectors each encoding one or more components of a composition or system described herein.
  • 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.
  • At least one of the three components, a nucleic acid encoding one or more effector proteins, one or more donor nucleic acids, and one or more guide nucleic acids or a nucleic acid encoding the one or more guide nucleic acids, or a variant thereof is provided in a different vector.
  • the nucleic acid encoding the effector protein, and a guide nucleic acid or a nucleic acid encoding the guide nucleic acid are provided in different vectors.
  • the donor nucleic acid is encoded by a different vector than the vector encoding the effector protein and the guide nucleic acid.
  • compositions and systems provided herein comprise a lipid particle.
  • a lipid particle is a lipid nanoparticle (LNP).
  • 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 (Kulkami 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.
  • lipids and polymers are cationic polymers, cationic lipids, ionizable lipids, or bio-responsive polymers.
  • the ionizable lipids exploits chemicalphysical 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.
  • the LNP comprises one or more of Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene- 1,3,5-tricarboxamide (TT3), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2- oleoylsn-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Choi), 1,2-dimyristoyl-sn-glycerol, and methoxypolyethylene glycol (DMG- PEChooo), derivatives, analogs, or variants thereof.
  • TT3 2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • POPE l-palmitoyl-2- oleoylsn-glycero-3-phosphoethanolamine
  • DSPC l,
  • 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 the effector protein, 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 effector protein, 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 effector protein 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 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.
  • 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.
  • 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 may be single-stranded or double stranded, linear or circular, segmented or non-segmented.
  • the nucleic acid may comprise 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 y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno- associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses.
  • retroviruses e.g., lentiviruses and y-retroviruses
  • adenoviruses e.g., lentiviruses and y-retroviruses
  • AAVs adeno-associated viruses
  • the viral vector is a recombinant viral vector.
  • the viral vector may be a retroviral vector.
  • Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In some embodiments, the viral vector is a recombinant viral vector.
  • gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome.
  • the viral vector is a chimeric
  • the viral vector corresponds to a virus of a specific serotype.
  • the viral vector is an AAV.
  • the AAV may be any AAV known in the art.
  • the viral vector corresponds to a virus of a specific serotype.
  • the 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, and an AAV12 serotype.
  • 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.
  • the AAV vector may be 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 may be 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 nucleotide sequence encoding genome editing tools.
  • the genome editing tools comprise a nucleic acid encoding one or more effector proteins, a nucleic acid encoding one or more fusion proteins (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 delivery vectors herein comprise packaging a nucleic acid encoding an effector protein and a guide nucleic acid, or a combination thereof, into an AAV vector.
  • 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 a Cas effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector.
  • promoters, staffer sequences, and any combination thereof may be packaged in the AAV vector.
  • the AAV vector can 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 may be not the same.
  • ITR inverted terminal repeat
  • the Rep gene and ITR from a first AAV serotype e.g., AAV2
  • a second AAV serotype e.g., AAV9
  • a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be 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. In some embodiments, rAAV is produced in an insect cell. In some embodiments, the insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells may comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells may comprise 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. In some embodiments, 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.
  • 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 the reagents.
  • the target nucleic acid is a double stranded nucleic acid.
  • the double stranded nucleic acid is DNA.
  • the target nucleic acid may be an RNA.
  • the target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non-coding RNA, long non-coding RNA, and microRNA (miRNA).
  • the target nucleic acid is complementary DNA (cDNA) synthesized from a singlestranded RNA template in a reaction catalyzed by a reverse transcriptase.
  • the target nucleic acid is single-stranded RNA (ssRNA) or mRNA.
  • the target nucleic acid is mRNA.
  • the target nucleic acid is from a virus, a parasite, or a bacterium described herein.
  • an effector protein or a multimeric complex thereof recognizes a PAM on a target nucleic acid.
  • multiple effector proteins of the multimeric complex recognize a PAM on a target nucleic acid.
  • only one effector protein of the multimeric complex recognizes a PAM on a target nucleic acid.
  • the PAM is 3’ to the spacer region of the guide nucleic acid (e.g., a crRNA or sgRNA) described herein.
  • the PAM is directly 3’ to the spacer region of the guide nucleic acid (e.g., a crRNA or sgRNA) described herein.
  • the PAM sequence comprises a nucleotide sequence listed in TABLE 3.
  • the 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%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of TABLE 1A and TABLE IB, and the target nucleic acid comprises a PAM sequence of any one of the nucleotide sequences as set forth in TABLE 3.
  • a target nucleic acid comprising a target sequence comprises a PAM 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 may be 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 an effector system described herein.
  • the 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 nucleosides. In some embodiments, the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 linked nucleosides. In some embodiments, 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 nucleosides.
  • 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 nucleosides.
  • a target nucleic acid comprises a portion or a specific region of a nucleic acid from a genomic locus from a gene described herein. Non-limiting examples of genes are recited in TABLE 10. Nucleic acid sequences of target nucleic acids and/or corresponding genes are readily available in public databases as known and used in the art.
  • the target nucleic acid is selected from TABLE 10.
  • the target nucleic acid comprises one or more target sequences.
  • the one or more target sequence is within any one of the target nucleic acids set forth in TABLE 10.
  • the target nucleic acid is a eukaryotic gene.
  • the target nucleic acid is a mammalian gene.
  • the target nucleic acid is a human gene.
  • the target nucleic acid sequence comprises a nucleotide sequence within a human safe harbor locus.
  • the human safe harbor locus comprises any one of: exon 1-2 of Angptl3 (human chromosome 1), exon 1-2 of AAVS1 (PPP1R12C) (human chromosome 19), exon 1-2 of ALB (human chromosome 4), exon 1-2 of ApoC3 (human chromosome 11), exon 1-2 of ASGR2 (human chromosome 17), exon 1-2 of CCR5 (human chromosome 3), exon 1-2 of FIX (F9) (human chromosome 10), exon 1-2 of Gys2 (human chromosome 12), exon 1-2 of HGD (human chromosome 3), exon 1-2 of Lp(a) (human chromosome 6), exon 1-2 of Pcsk9 (human chromosome 1), exon 1-2 of Serpinal (human chromosome 14), exon 1-2 of TF (human chromosome 3), and exon 1-2 of TTR (human chromosome
  • the human safe harbor locus comprises at least one sequence in AAVS 1, CCR5, human ortholog sequence of the mouse Rosa26, or human albumin (also referred to as human serum albumin or HSA or ALB). In some instances, the human safe harbor locus is located in human chromosome 2 or human chromosome 4. In some embodiments, 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.
  • 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 the nucleotide sequence recited in TABLE 10.
  • At least a portion of the target nucleic acid that a guide nucleic acid binds is within a nucleotide sequence about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 55 or more, about 60 or more, about 65 or more, about 70 or more, about 75 or more, about 80 or more, about 85 or more, about 90 or more, about 95 or more, about 100 or more, about 105 or more, about 110 or more, about 115 or more, about 120 or more, about 125 or more, about 130 or more, about 135 or more, about 140 or more, about 145 or more, or about 150 or more nucleotides adjacent to: the start of a targeted intron, the end of a targeted intron, or both.
  • the guide nucleic acid comprises the spacer 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 SEQ ID NO: 301-510, wherein the effector protein has 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 SEQ ID NO: 6-12, and SEQ ID NO: 228-230.
  • the guide nucleic acid comprises the spacer 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 SEQ ID NO: 511-535, wherein the effector protein has 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 SEQ ID NO: 13.
  • the guide nucleic acid comprises the spacer 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 SEQ ID NO: 536-575, wherein the effector protein has 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 SEQ ID NO: 47.
  • the guide nucleic acid comprises the spacer 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 SEQ ID NO: 576-604, wherein the effector protein has 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 SEQ ID NO: 231.
  • the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a spacer sequence and/or a repeat sequence, wherein the spacer sequence 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 4, wherein the repeat sequence 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 6, and wherein the spacer sequence hybridizes to a target 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: 266
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 47; a crRNA comprising a repeat 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: 254, and a spacer 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 SEQ ID NO: 536-575, wherein the spacer sequence hybridizes to a target 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: 266.
  • compositions disclosed herein comprises an effector protein comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 231; a crRNA comprising a repeat 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: 255 or 1789, and a spacer 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 SEQ ID NO: 576-604, wherein the spacer sequence hybridizes to a target 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: 266.
  • the guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a spacer sequence and/or a handle sequence, wherein the spacer sequence 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 4, wherein the handle sequence 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 7, and wherein the spacer sequence hybridizes to a target 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: 266.
  • the handle sequence comprises one or more of an intermediary sequence, a repeat sequence, and a linker.
  • compositions disclosed herein comprises 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%, at least 99%, or 100% identical to SEQ ID NO: 13; a guide nucleic acid comprising 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%, or 100% identical to any one of SEQ ID NO: 259-261, and a spacer 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 SEQ ID NO: 511-535, wherein the spacer sequence hybridizes to a target 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: 266
  • compositions disclosed herein comprises 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%, at least 99%, or 100% identical to SEQ ID NO: 231; a guide nucleic acid comprising 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%, or 100% identical to any one of SEQ ID NO: 262-264, and a spacer 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 SEQ ID NO: 576-604, wherein the spacer sequence hybridizes to a target 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: 266
  • a guide nucleic acid or a nucleic acid encoding the guide nucleic acid comprises a spacer sequence, wherein the spacer sequence hybridizes to a target 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: 266.
  • the spacer sequence 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 4.
  • the target sequence comprises at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 10, a complement thereof, or a reverse complement thereof.
  • 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 the nucleotide sequence recited in TABLE 10.
  • 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 any one of the nucleotide sequences recited in TABLE 10.
  • 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 of any one of the nucleotide sequences recited in TABLE 10.
  • the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 linked nucleotides of any one of the nucleotide sequences recited in TABLE 10.
  • 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 of any one of the nucleotide sequence recited in TABLE 10.
  • 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 of any one of the nucleotide sequences recited in TABLE 10.
  • compositions, systems, and methods described herein comprise a modified target nucleic acid which can describe a target nucleic acid wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein.
  • the modification is an alteration in the sequence of the target nucleic acid.
  • the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target nucleic acid.
  • the modification is a mutation.
  • the target nucleic acid is in a cell.
  • the cell is a hepatocyte.
  • the cell is a human cell.
  • the human cell is a hepatocyte, stem cell, progenitor cell, induced pluripotent stem cell (iPSC), or a cell derived from an iPSC cell.
  • a donor nucleic acid comprises a nucleic acid that is incorporated into a target nucleic acid or target sequence.
  • 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
  • transgenes described herein can be inserted or integrated into a target nucleic acid.
  • the donor nucleic acid comprises a human gene.
  • the donor nucleic acid comprises a gene encoding a human functional protein.
  • Functional human proteins as described herein include proteins that are encoded by transgenes incorporated in donor nucleic acids described herein. Specific examples of functional human proteins include wildtype and engineered versions of proteins that maybe deficient, under-expressed or aberrantly expressed in certain human subjects. In some instances, these subjects suffer from disorders that result in deficiency, or aberrant expression of these proteins. Compositions and methods provided herein are useful to treat conditions characterized by such deficiency, or aberrant expression.
  • a compositions and systems comprise a donor nucleic acid encoding a functional protein that is poorly expressed in subjects with genetic disorders such as monogenic disorders.
  • a functional protein 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.
  • functional human proteins that are poorly expressed in subjects with genetic disorders include CFTR, DMD, Al AT, GAA, FXN, F8, F9, SOD1, C9, HTT, MECP2, SMN1, TARDBP, FUS, RHO, and USH2A.
  • Exemplary amino acid sequences of functional human proteins are provided in TABLE 11.
  • a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • a point mutation optionally comprises a substitution, insertion, or deletion.
  • a mutation comprises a chromosomal mutation.
  • a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation.
  • a mutation comprises a copy number variation.
  • a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. The mutation may be located in a non-coding region or a coding region of a gene.
  • a donor nucleic acid may be inserted at 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 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 wildtype.
  • the wildtype protein comprises human wildtype protein sequence.
  • the human protein comprises an amino acid sequence recited in TABLE 11.
  • the donor nucleic acid encoding amino acid 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 amino acid sequences recited in TABLE 11.
  • methods comprise contacting a target nucleic acid with 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB, thereby introducing a single-stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein, optionally 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%, at least 97%, at least 98%, at least 99% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB, to generate a second cleavage site in the target nucleic acid, ligating the regions flanking the first and second cleavage site,
  • compositions comprising one or more effector proteins described herein or nucleic acids encoding the one or more effector proteins, one or more guide nucleic acids described herein or nucleic acids encoding the one or more guide nucleic acids described herein, or combinations thereof.
  • one or more of a repeat sequence, a handle sequence, and intermediary sequence of the one or more guide nucleic acids are capable of interacting 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 comprising: an effector protein, or a nucleic acid encoding the effector protein; a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and a donor nucleic acid.
  • 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 recited in TABLE 1A and TABLE IB.
  • the guide nucleic acid is a crRNA.
  • the crRNA comprises a spacer sequence and a repeat sequence.
  • the guide nucleic acid is a single guide RNA (sgRNA).
  • the sgRNA comprises a spacer sequence and a handle sequence.
  • the handle sequence comprises one or more of an intermediary sequence, a repeat sequence, and a linker.
  • the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid.
  • the target sequence comprises a nucleotide sequence within a human safe harbor locus.
  • the target sequence comprises any one of the nucleotide sequences recited in TABLE 10.
  • the donor nucleic acid encodes a transgene that comprises a functional human protein that is expressed upon incorporation into the human safe harbor locus.
  • 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. In some embodiments, compositions described herein comprise liposomes (e.g., cationic lipids or neutral lipids), dendrimers, lipid nanoparticle (LNP), or cellpenetrating peptides. In some embodiments, compositions described herein comprise an LNP.
  • compositions and Modes of Administration are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, fusion effector proteins, guide nucleic acids, or donor nucleic acid as described herein and any combination thereof. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.
  • compositions including pharmaceutical compositions, comprise a viral vector encoding a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion effector protein.
  • compositions comprise a virus comprising a viral vector encoding a fusion effector protein, an effector protein, a fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • the virus may be a lentivirus.
  • the virus may be an adenovirus.
  • the virus may be a non-replicating virus.
  • the virus may be an adeno- associated virus (AAV).
  • 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.
  • compositions are in the form of a solution (e.g. , a liquid) .
  • the solution may be 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.
  • compositions comprise an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutical compositions comprise one or more nucleic acids encoding an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • guide nucleic acid can be a plurality of guide nucleic 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 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences of TABLE 1A and TABLE IB.
  • compositions comprise an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutical compositions comprise one or more nucleic acids encoding an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.
  • guide nucleic acid can be a plurality of guide nucleic 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 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence recited in TABLE 1A and TABLE IB.
  • the nucleotide sequence of the gRNA 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 gRNA sequences recited in TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • the nucleotide sequence of the gRNA is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1825, 1828, 1846 or 1847.
  • the guide nucleic acid comprises a nucleotide sequence of SEQ ID NO: 1825, 1828, 1846 or 1847.
  • the nucleotide sequence of the gRNA 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 gRNA sequences recited in TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • the nucleotide sequence of the gRNA is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1825, 1828, 1846 or 1847.
  • systems for modifying, or editing a target nucleic acid comprising the effector proteins or nucleic acids encoding the effector proteins described herein, or a multimeric complex thereof.
  • Systems may be used to modify or edit a target nucleic acid.
  • Systems may be used to insert 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 amino acid sequences of TABLE 1A and TABLE IB.
  • the amino acid sequence of the effector protein 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 amino acid sequences of TABLE 1A and TABLE IB.
  • the guide nucleic acid comprises at least one nucleotide sequence selected from the nucleotide sequences in any one of TABLE 6 and TABLE 5
  • systems comprise an effector protein described herein, a guide nucleic acid described herein, a reagent, support medium, or a combination thereof.
  • 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 amino acid sequences of TABLE 1A and TABLE IB.
  • the amino acid sequence of the effector protein 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 amino acid sequences of TABLE 1A and TABLE IB.
  • 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 the nucleotide sequence set forth in TABLE 6.
  • 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) a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and (iii) 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 effector protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of amino acid sequences recited in TABLE 1A and TABLE IB.
  • the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid.
  • the target sequence comprises a nucleotide sequence within the safe harbor.
  • the safe harbor comprises at least 90% sequence identity to any one of the nucleotide sequences recited in TABLE 10.
  • the guide nucleic acid comprises at least one 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 the nucleotide sequence of any one of TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • the guide nucleic acid comprises at least one 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: 1825, 1828, 1846 or 1847.
  • 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 a nucleotide sequence recited in TABLE 10.
  • the donor nucleic acid comprises a nucleotide sequence encoding 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 the amino acid sequence recited in TABLE 11.
  • systems comprise an effector protein described herein, a guide nucleic acid described herein, a reagent, support medium, or a combination thereof.
  • 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 the amino acid sequence recited in TABLE 1A and TABLE IB.
  • the amino acid sequence of the effector protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence recited in TABLE 1A and TABLE IB.
  • 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 and TABLE 5 and 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 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 and TABLE 5 and 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 7.
  • the nucleotide sequence of the guide nucleic acid is 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% identical to any one of the gRNA sequences recited in TABLE 9.
  • systems described herein comprise separate compositions, solutions, containers, kits, vectors, or the like that individually comprise an effector protein, a nucleic acid encoding the effector protein, a guide nucleic acid, a nucleic acid encoding the guide nucleic acid, donor nucleic acid, or a combination thereof.
  • Such systems can provide for separate delivery of the effector protein, the nucleic acid encoding the effector protein, the guide nucleic acid, the nucleic acid encoding the guide nucleic acid, or the donor nucleic acid described herein.
  • systems described herein comprise a composition, solution, container, kit, vector, or the like that comprises two or more of an effector protein, a nucleic acid encoding the effector protein, a guide nucleic acid, a nucleic acid encoding the guide nucleic acid, and donor nucleic acid described herein.
  • Such systems can provide for delivery of two or more of the effector protein, the nucleic acid encoding the effector protein, the guide nucleic acid, the nucleic acid encoding the guide nucleic acid, and the donor 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.
  • any two system components are provided in different solutions or containers.
  • Suitable containers include, for example, test wells, bottles, vials, 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.
  • a system may include labels listing contents and/or instructions for use, or package inserts with instructions for use.
  • a set of instructions will also typically be included.
  • a label is on or associated with the container.
  • a 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.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
  • 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 may improve 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.
  • 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 recombina
  • systems comprise a PCRtube, a PCR well or a PCR plate.
  • the wells of the PCR plate may be 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 may be pre-aliquoted with a guide nucleic acid targeting a target sequence, an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence.
  • systems comprise a support medium; a guide nucleic acid targeting a target sequence; and an effector protein capable of being 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 for modifying a target nucleic acid comprises a PCR plate; a guide nucleic acid targeting a target sequence; and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence.
  • the wells of the PCR plate may be pre- aliquoted with the guide nucleic acid targeting a target sequence, and an effector protein capable of being activated when complexed with the guide nucleic acid and the target sequence. A user may thus add 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. Sometimes, the nucleic acid amplification is performed for 1 to 60, 5 to 55, 10 to 50, 15 to 45, 20 to 40, or 25 to 35 minutes. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. In some embodiments, the nucleic acid amplification reaction is performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, or any value 20 °C to 45 °C.
  • the nucleic acid amplification reaction is performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, or 45°C, or any value 20 °C to 45 °C. In some embodiments, the nucleic acid amplification reaction is performed at a temperature of 20°C to 45°C, 25°C to 40°C, 30°C to 40°C, or 35°C to 40°C.
  • the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH is less than 7. In some embodiments, the pH is greater than 7.
  • a host may be any suitable host, such as a host cell.
  • a host cell may be 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, which eukaryotic or prokaryotic cells may be, or have been, used as recipients for methods of introduction described herein, and include the progeny of the original cell which has been transformed by the methods of introduction described herein.
  • a host cell may be 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, which eukaryotic or prokaryotic cells may be, or have been, used as recipients for methods of introduction described herein,
  • a host cell may be 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.
  • the nucleic acid and/or protein are introduced into a disease cell comprised in a pharmaceutical composition comprising the guide nucleic acid and/or effector protein and a pharmaceutically acceptable excipient.
  • molecules of interest such as nucleic acids of interest
  • polypeptides such as an effector protein
  • vectors such as lipid particles and/or viral vectors may be introduced to a host. Introduction may be 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 an effector protein, a nucleic acid that, when transcribed, produces an engineered guide nucleic acid, and/or a donor 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. Further methods are described throughout.
  • PEI polyethyleneimine
  • Introducing one or more nucleic acids into a host cell may occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing one or more nucleic acids into a host cell may be carried out in vivo or ex vivo. Introducing one or more nucleic acids into a host cell may be carried out in vitro.
  • an effector protein may be provided as RNA.
  • the RNA may be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the effector protein).
  • the RNA may be 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 may be 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 may be introduced directly to a host.
  • host cells may be 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 may also be introduced directly to a host.
  • an engineered guide nucleic acid may be 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) described herein may also be introduced directly to a host.
  • polypeptides described herein may be modified to promote introduction to a host.
  • polypeptides described herein may be modified to increase the solubility of the polypeptide.
  • Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility.
  • the domain may be linked to the polypeptide through a defined protease cleavage site, such as TEV sequence which is cleaved by TEV protease.
  • the linker may also include 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 may be modified to improve stability.
  • the polypeptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.
  • Polypeptides may also be modified to promote uptake by a host, such as a host cell.
  • a polypeptide described herein may be 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.
  • a permeant peptide may be 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 may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site may be determined by suitable methods.
  • formulations of introducing compositions or components of a system described herein to a host comprise an effector protein and a carrier (e.g., excipient, diluent, vehicle, or filling agent).
  • a carrier e.g., excipient, diluent, vehicle, or filling agent.
  • the effector protein is provided in a pharmaceutical composition comprising the effector protein and any pharmaceutically acceptable excipient, carrier, or diluent.
  • editing refers to modifying the nucleotide sequence of a target nucleic acid.
  • compositions and systems disclosed herein may also be capable of making epigenetic modifications of target nucleic acids. Effector proteins, multimeric complexes thereof and systems described herein may be used for editing or modifying a target nucleic acid.
  • Editing a target nucleic acid may comprise 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 modifying (e.g., methylating, demethylating, deaminating, or oxidizing) of one or more nucleotides of the target nucleic acid.
  • Methods of editing may comprise contacting a target nucleic acid with an effector protein described herein and a guide nucleic acid, wherein the 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the amino acid sequences set forth in TABLE 1A and TABLE IB.
  • 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 the nucleotide sequence set forth in TABLE 6.
  • 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 and TABLE 5 and 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 the nucleotide sequence recited in TABLE 6.
  • 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 and TABLE 5 and 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 7.
  • the nucleotide sequence of the guide nucleic acid is 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% identical to any one of the nucleotide sequences recited in TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • the nucleotide sequence of the guide nucleic acid is 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% identical to any one of SEQ ID NO: 1825, 1828, 1846 or 1847.
  • the compositions, methods or systems comprise a nucleic acid expression vector, or use thereof, to introduce an effector protein, 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.
  • AAV adeno associated viral
  • 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 a Cas protein, guide nucleic acid, donor template or any combination thereof to a cell.
  • Non-limiting examples of 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 editing may comprise contacting a target nucleic acid with one or more components, compositions or systems described herein.
  • a method of editing comprises contacting a target nucleic acid with at least one of: a) one or more effector proteins, or one or more nucleic acids encoding one or more effector proteins; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids.
  • a method of editing 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 effector proteins, or one or more nucleic acids encoding one or more effector proteins; or b) one or more guide nucleic acids, or one or more nucleic acids encoding one or more guide nucleic acids.
  • Editing may introduce a mutation (e. g. , point mutations, insertions, deletions) in a target nucleic acid relative to a corresponding wildtype nucleotide sequence. Editing may remove or insert a nucleic acid sequence to produce a corresponding wildtype protein. Editing may remove/insert tissue-specific nucleic acid sequence in a target nucleic acid. Editing may be used to generate gene knock-in, gene editing, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a cell.
  • a mutation e. g. , point mutations, insertions, deletions
  • Editing may remove or insert a nucleic acid sequence to produce a corresponding wildtype protein. Editing may remove/insert tissue-specific nucleic acid sequence in a target nucleic acid. Editing may be used to generate gene knock-in, gene editing, or a combination thereof. Methods of the disclosure may be targeted to any locus in a genome of a
  • Editing may comprise 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
  • the effector proteins introduce a single -stranded break in a target nucleic acid to produce a cleaved nucleic acid.
  • the effector protein is capable of introducing a break in a single stranded RNA (ssRNA).
  • the effector protein may be 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).
  • HDR homology directed repair
  • NHEJ non-homologous end joining
  • a double-stranded break in the target nucleic acid may be 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 percentage is based on 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.
  • methods of editing 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 may be 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.
  • compositions, systems, and methods described herein can 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 can be edited by the compositions, systems, and methods described herein.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides can be edited by the compositions, systems, and methods described herein.
  • 10, 20, 30, 40, 50, 60, 70, 80 90, 100 or more nucleotides, or any integer in between can be 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 can be edited by the compositions, systems, and methods described herein.
  • Methods may comprise use of two or more effector proteins.
  • 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 effector protein, wherein the effector protein comprises at least 75% sequence identity to the amino acid sequence of TABLE 1A and TABLE IB; and (b) a second engineered guide nucleic acid comprising a region that binds to a second effector protein, wherein the effector protein comprises at least 75% sequence identity to the amino acid sequence of TABLE 1A and TABLE IB, wherein the first engineered guide nucleic acid comprises an additional region that binds to the target nucleic acid and wherein the second engineered guide nucleic acid comprises an additional region that binds to the target nucleic acid.
  • the guide nucleic acid comprises a crRNA sequence comprising 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 the nucleotide sequence of TABLE 6.
  • modifying a target nucleic acid may comprise deleting a sequence from a target nucleic acid. In some embodiments, modifying a target nucleic acid may comprise replacing a sequence in a target nucleic acid with a second sequence. In some embodiments, modifying a target nucleic acid may comprise introducing a sequence into a target nucleic acid. For example, a beneficial sequence or a sequence that may reduce or eliminate a disease may be inserted into the target nucleic acid. In some embodiments, the beneficial sequence 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 an amino acid sequence recited in TABLE 11.
  • methods comprise editing a target nucleic acid with two or more effector proteins.
  • Editing a target nucleic acid may comprise introducing a two or more single -stranded breaks in a target nucleic acid.
  • a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid.
  • the guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting 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 may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid.
  • modifying a target nucleic acid may comprise 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.
  • modifying a target nucleic acid may 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 programmable nickase and hybridizes to a second region of the target nucleic acid.
  • the first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid
  • the second effector protein may introduce 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 may be removed, thereby modifying the target nucleic acid.
  • a segment of the target nucleic acid between the first break and the second break may be replaced (e.g., with donor nucleic acid), thereby modifying the target nucleic acid.
  • 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 of TABLE 1A and TABLE IB
  • methods comprise inserting a donor nucleic acid into a cleaved target nucleic acid.
  • the donor nucleic acid may be inserted at a specified (e.g. , effector protein targeted) point within the target nucleic acid.
  • methods comprise contacting a target nucleic acid with an effector protein comprising an amino acid sequence that is 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 the amino acid sequence of TABLE 1A and TABLE IB, thereby introducing a single -stranded break in the target nucleic acid; contacting the target nucleic acid with a second effector protein, optionally comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of TABLE 1A and TABLE IB, 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 ex
  • methods comprise editing a target nucleic acid with two or more effector proteins.
  • Editing a target nucleic acid may comprise introducing a two or more single-stranded breaks in a target nucleic acid.
  • a break may be introduced by contacting a target nucleic acid with an effector protein and a guide nucleic acid.
  • the guide nucleic acid may bind to the effector protein and hybridize to a region of the target nucleic acid, thereby recruiting 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 may activate the effector protein, and the effector protein may introduce a break (e.g., a single stranded break) in the region of the target nucleic acid.
  • modifying a target nucleic acid may comprise 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.
  • modifying a target nucleic acid may 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 programmable nickase and hybridizes to a second region of the target nucleic acid.
  • the first effector protein may introduce a first break in a first strand at the first region of the target nucleic acid
  • the second effector protein may introduce 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 may be removed, thereby modifying the target nucleic acid.
  • a segment of the target nucleic acid between the first break and the second break may be replaced (e.g. , with donor nucleic acid), thereby modifying the target nucleic acid.
  • the 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%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of TABLE 1A and TABLE IB.
  • the guide nucleic acid comprises a crRNA sequence comprising 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 the nucleotide sequence of TABLE 6.
  • editing is achieved by fusing an effector protein to a heterologous sequence.
  • the heterologous sequence may be a suitable fusion partner, e.g., a protein that provides recombinase activity by acting on the target nucleic acid.
  • the fusion protein comprises an effector protein fused to a heterologous sequence by a linker.
  • the heterologous sequence or fusion partner may be a base editing domain.
  • the base editing domain may be an ADAR1/2 or any functional variant thereof.
  • the heterologous sequence or fusion partner may be fused to the C-terminus, N-terminus, or an internal portion (e.g., a portion other than the N- or C-terminus) of the effector protein.
  • the heterologous sequence or fusion partner may be fused to the effector protein by a linker.
  • a linker may be a peptide linker or a non-peptide linker.
  • the linker is an XTEN linker.
  • the linker comprises one or more repeats a tri -peptide GGS.
  • 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.
  • a non-peptide linker may be 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
  • editing or modification of a target nucleic acid can be locus specific, wherein compositions, systems, and methods described herein can edit or modify a target nucleic acid at one or more specific loci to effect one or more specific mutations comprising sequence deletion, sequence knock-in, or any combination thereof.
  • editing or modification of a specific locus can effect sequence knock-in.
  • sequence knock-in is a modification where one or more sequences is inserted into a target nucleic acid relative to a target nucleic acid without the sequence knock-in.
  • editing or modification of a specific locus can effect sequence knock-in and sequence deletion.
  • editing or modification of a target nucleic acid can be locus specific, modification specific, or both. In certain embodiments, editing or modification of a target nucleic acid can be locus specific, modification specific, or both, wherein compositions, systems, and methods described herein comprise an effector protein described herein, and a guide nucleic acid described herein. In certain embodiments, edition or modification of a target nucleic acid is specific to intron 1 of mammalian albumin gene.
  • Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vivo.
  • Methods of editing a target nucleic acid or modulating the expression of a target nucleic acid may be performed in vitro.
  • a plasmid may be modified 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 may be performed ex vivo.
  • methods may comprise obtaining a cell from a subject, modifying a target nucleic acid in the cell with methods described herein, and returning the cell to the subject.
  • the term donor nucleic acid refers to a sequence of nucleotides that will be or has been inserted at the site of cleavage by the effector protein (cleaving (hydrolysis of a phosphodiester bond) of a nucleic acid resulting in a nick or double strand break -nuclease activity).
  • the term donor nucleic acid refers to a sequence of DNA that serves as a template in the process of homologous recombination, which may carry the modification that is to be or has been introduced into the target nucleic acid. By using this donor nucleic acid as a template, the genetic information, including the modification, is copied into the target nucleic acid by way of homologous recombination.
  • Donor nucleic acids of any suitable size may be integrated into a target nucleic acid or genome.
  • the donor polynucleotide integrated into a 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.
  • donor nucleic acids are more than 500 kilobases (kb) in length.
  • the donor nucleic acid may comprise a sequence that is derived from an animal.
  • the animal may be human.
  • the animal may be a non-human animal, such as, by way of non-limiting example, a mouse, rat, hamster, rabbit, pig, bovine, deer, sheep, goat, chicken, cat, dog, ferret, a bird, non-human primate (e.g., marmoset, rhesus monkey).
  • the non-human animal may be a domesticated mammal or an agricultural mammal.
  • 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 may carry a modification that is to be or has been introduced into a target nucleic acid.
  • the genetic information including the modification, is copied into the target nucleic acid by way of homologous recombination.
  • the target nucleic acid upon modification of a target nucleic acid by compositions, systems, and methods described herein, can comprise an intron deletion, intron knock-in, or a combination thereof.
  • Methods may comprise contacting a cell with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding an effector protein, wherein the 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%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences of TABLE 1A and TABLE IB
  • a nucleic acid e.g., a plasmid or mRNA
  • Methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding an effector protein, a guide nucleic acid (e.g., a crRNA or sgRNA), a donor nucleic acid or any combination thereof.
  • a nucleic acid e.g., a plasmid or mRNA
  • Methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding a guide nucleic acid, a tracrRNA, a crRNA, or any combination thereof.
  • Contacting may comprise 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.
  • nanoparticle delivery e.g., lipid or gold nanoparticle delivery
  • CPP cell-penetrating peptide
  • Methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding an effector protein, a guide nucleic acid (e.g., a crRNA or sgRNA), a donor nucleic acid or any combination thereof.
  • methods may comprise contacting cells with a nucleic acid (e.g., a plasmid or mRNA) comprising a nucleotide sequence encoding a guide nucleic acid.
  • the nucleotide sequence of the guide nucleic acid is 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% identical to of any one of the gRNA sequences recited in TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 8, and TABLE 9.
  • the guide nucleic acid comprises a crRNA sequence comprising 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the spacer sequences recited in TABLE 4 and TABLE 5, and 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the repeat sequences recited in TABLE 6.
  • the guide nucleic acid comprises a crRNA sequence comprising 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the spacer sequences recited in TABLE 4 and TABLE 5, and 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%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the handle sequences recited in TABLE 7.
  • Contacting may comprise 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.
  • nanoparticle delivery e.g., lipid or gold nanoparticle delivery
  • CPP cell-penetrating peptide
  • Methods may comprise contacting a cell with an effector protein or a multimeric complex thereof, wherein the 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%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences of TABLE 1A and TABLE IB.
  • compositions of the disclosure may be administered to a subject.
  • a subject may be a human.
  • a subject may be a mammal (e.g. , rat, mouse, cow, dog, pig, sheep, horse).
  • a subject may be a vertebrate or an invertebrate.
  • a subject may be a laboratory animal.
  • a subject may be a patient.
  • a subject may be at risk of developing, suffering from, or displaying symptoms of disease.
  • a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • a point mutation optionally comprises a substitution, insertion, or deletion.
  • a mutation comprises a chromosomal mutation.
  • a chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation.
  • a mutation comprises a copy number variation.
  • a copy number variation can comprise a gene amplification or an expanding trinucleotide repeat.
  • the disease or disorder comprises Pompe disease, glycogen storage disorder, cystic fibrosis, muscular dystrophy, Freidreich’s ataxia, amylotrophic lateral scleorsis, hemophilia, Huntington’s disease, retinal dystrophy, Rett syndrome, sickle cell disease, or a combination thereof.
  • the method for treating is performed in a cell. In some embodiments, the method for treating is performed in vivo. In some embodiments, the cell is 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
  • a Cas protein is used to effect the insertion.
  • Cas proteins may be fused to transcription activators or transcriptional repressors or deaminases or other nucleic acid modifying proteins.
  • Cas proteins need not be fused to a partner protein to accomplish the required protein (expression) modification.
  • treatment of a disease comprises administration of a gene therapy.
  • Gene therapy comprises use of a recombinant nucleic acid (DNA or RNA), administered for the purpose to add a gene sequence.
  • a gene therapy 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. Methods of gene therapy 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.
  • a transgene is 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.
  • a gene inserted for treatment comprises a nucleotide sequence that encodes wild-type functional human protein or a variant thereof.
  • the mutated donor nucleic acid encodes a function human protein.
  • compositions 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 comprise administering a composition or cell described herein to a subject.
  • the disease may be a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof.
  • the disease may be an inherited disorder, also referred to as a genetic disorder.
  • the disease may be the result of an infection or associated with an infection.
  • methods of treating a disease in a subject comprises administering to the subject the composition described herein, or the components of the systems described herein. In some embodiments, at least two components of the system are administered separately. In some embodiments, at least two components of the system are administered simultaneously. In some embodiments, the subject treated by the methods described herein has any one of the diseases described herein. In some embodiments, administering to the subject the composition described herein, or the components of the systems described herein, increases the amount of the functional human protein in the subject (e.g., in a biological sample from the subject) as compared to the subject before administration.
  • compositions and methods described herein may be used to treat, prevent, or inhibit a disease or syndrome in a subject.
  • the disease is a liver disease, a lung disease, an eye disease, or a muscle disease.
  • Exemplary diseases and syndromes include but are not limited to the diseases and syndromes listed in TABLE 12.
  • compositions and methods edit at least one gene associated with a disease described herein or the expression thereof.
  • the disease is Alzheimer’s disease and the gene is selected from APP, BACE-1, PSD95, MAPT, PSENI, PSEN2, and APOEe4.
  • the disease is Parkinson’s disease and the gene is selected from SNCA, GDNF, and LRRK2.
  • the disease comprises Centronuclear myopathy and the gene is DNM2.
  • the disease is Huntington's disease and the gene is HTT.
  • the disease is Alpha- 1 antitrypsin deficiency (AATD) and the gene is SERPINA1.
  • the disease is amyotrophic lateral sclerosis (ALS) and the gene is selected from SOD1, FUS, C9ORF72, ATXN2, TARDBP, and CHCHD10.
  • the disease comprises Alexander Disease and the gene is GFAP.
  • the disease comprises Angelman Syndrome and the gene is UBE3A.
  • the disease comprises calcific aortic stenosis and the gene is Apo(a).
  • the disease comprises CD3Z-associated primary T-cell immunodeficiency and the gene is CD3Z or CD247.
  • the disease comprises CD 18 deficiency and the gene is ITGB2.
  • the disease comprises CD40L deficiency and the gene is CD40L.
  • the disease is congenital adrenal hyperplasia and the gene is CAH1.
  • the disease comprises CNS trauma and the gene is VEGF.
  • the disease comprises coronary heart disease and the gene is selected from FGA, FGB, and FGG.
  • the disease comprises MECP2 Duplication syndrome and Rett syndrome and the gene is MECP2.
  • the disease comprises a bleeding disorder (coagulation) and the gene is FXI.
  • the disease comprises fragile X syndrome and the gene is FMRI .
  • the disease is Retinitis pigmentosa and the gene is selected from PDE6B, RHO, RP1, RP2, RPGR, PRPH2, IMPDH1, PRPF31, CRB1, PRPF8, TULP1, CA4, HPRPF3, ABCA4, EYS, CERKL, FSCN2, TOPORS, SNRNP200, PRCD, NR2E3, MERTK, USH2A, PR0M1, KLHL7, CNGB1, TTC8, ARL6, DHDDS, BEST1, LRAT, SPARA7, CRX, CLRN1, RPE65, and WDR19.
  • the disease comprises Leber Congenital Amaurosis Type 10 and the gene is CEP 290.
  • the disease is cardiovascular disease and/or lipodystrophies and the gene is selected from ABCG5, ABCG8, AGP, ANGPTL3, APOCIII, APOA1, APOL1, ARH, CDKN2B, CFB, CXCL12, FXI, FXII, GATA-4, MIA3, MKL2, MTHFD1L, MYH7, NKX2-5, NOTCH1, PKK, PCSK9, PSRCI, SMAD3, and TTR.
  • the disease comprises acromegaly and the gene is GHR.
  • the disease is diabetes and the gene is GCGR.
  • the disease is NAFLD/NASH and the gene is selected from DGAT2 and PNPLA3.
  • the disease is cystic fibrosis and the gene is CFTR.
  • the disease is Duchenne muscular dystrophy and the gene is DMD.
  • the disease is ornithine transcarbamylase deficiency and the gene is OTC.
  • the disease comprises angioedema and the gene is PKK.
  • the disease comprises thalassemia and the gene is TMPRSS6.
  • the disease comprises achondroplasia and the gene is FGFR3.
  • the disease comprises Cri du chat syndrome and the gene is selected from CTNND2.
  • the disease comprises sickle cell anemia and the gene is Beta globin gene.
  • the disease comprises Alagille Syndrome and the gene is selected from JAG1 and NOTCH2.
  • the disease comprises Charcot-Marie-Tooth disease and the gene is selected from PMP22 and MFN2.
  • the disease comprises Crouzon syndrome and the gene is selected from FGFR2, FGFR3, and FGFR3.
  • the disease comprises Dravet Syndrome and the gene is selected from SCN1A and SCN2A.
  • the disease comprises Emery-Dreifuss syndrome and the gene is selected from EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43.
  • the disease comprises Factor V Leiden thrombophilia and the gene is F5.
  • the disease is fabry disease and the gene is GLA.
  • the disease is facioscapulohumeral muscular dystrophy and the gene is FSHD1.
  • the disease comprises Fanconi anemia and the gene is selected from FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCE FANCM, FANCN, FANCP, FANCS, RAD51C, and XPF.
  • the disease comprises Familial Creutzfeld-Jakob disease and the gene is PRNP.
  • the disease comprises Familial Mediterranean Fever and the gene is MEFV. In some embodiments, the disease comprises Friedreich's ataxia and the gene is FXN. In some embodiments, the disease comprises Gaucher disease and the gene is GBA. In some embodiments, the disease comprises hemochromatosis and the gene is HFE, optionally comprising a C282Y mutation. In some embodiments, the disease comprises Hemophilia A and the gene is FVIII. In some embodiments, the disease is hereditary angioedema and the gene is SERPING1. In some embodiments, the disease comprises histiocytosis and the gene is CD1. In some embodiments, the disease comprises immunodeficiency 17 and the gene is CD3D.
  • the disease comprises immunodeficiency 13 and the gene is CD4. In some embodiments, the disease comprises Common Variable Immunodeficiency and the gene is selected from CD19 and CD81. In some embodiments, the disease comprises Joubert syndrome and the gene is selected from INPP5E, TMEM216, AHI1, NPHP1, CEP 290, TMEM67, RPGRIP1L, ARL13B, CC2D2A, OFD1, TMEM138, TCTN3, ZNF423, and AMRC9. In some embodiments, the disease comprises leukocyte adhesion deficiency and the gene is CD18. In some embodiments, the disease comprises Li-Fraumeni syndrome and the gene is TP53.
  • the disease comprises Lynch syndrome and the gene is selected from MSH2, MLH1, MSH6, PMS2, PMS1, TGFBR2, and MLH3.
  • the disease comprises Marfan syndrome and the gene is FBN1.
  • the disease comprises mastocytosis and the gene is CD2.
  • the disease comprises methylmalonic acidemia and the gene is selected from MMAA, MMAB, andMUT.
  • the disease is myotonic dystrophy and the gene is selected from CNBP and DMPK.
  • the disease comprises neurofibromatosis and the gene is selected from NF1, and NF2.
  • the disease comprises osteogenesis imperfecta and the gene is selected from COL1A1, COL1A2, and IFITM5.
  • the disease comprises Peutz-Jeghers syndrome and the gene is STK11.
  • the disease comprises polycystic kidney disease and the gene is selected from PKD1 and PKD2.
  • the disease comprises Severe Combined Immune Deficiency and the gene is selected from IL7R, RAG1, and JAK3.
  • the disease comprises PRKAG2 cardiac syndrome and the gene is PRKAG2.
  • the disease comprises spinocerebellar ataxia and the gene is selected from ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, ATXN8OS, ATXN10, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND3, and FGF14.
  • the disease is thrombophilia due to antithrombin III deficiency and the gene is SERPINC1.
  • the disease is spinal muscular atrophy and the gene is SMNE
  • the disease comprises Usher Syndrome and the gene is selected from MY07A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, DFNB31, and CLRNE
  • the disease comprises von Willebrand disease and the gene is VWF.
  • the disease comprises Waardenburg syndrome and the gene is selected from PAX3, MITF, WS2B, WS2C, SNAI2, EDNRB, EDN3, and SOX10.
  • the disease comprises Wiskott-Aldrich Syndrome and the gene is WAS.
  • the disease comprises von Hippel-Lindau disease and the gene is VHP.
  • the disease comprises Wilson disease and the gene is ATP7B.
  • the disease comprises Zellweger syndrome and the gene is selected from PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.
  • the disease comprises infantile myofibromatosis and the gene is CD34.
  • the disease comprises platelet glycoprotein IV deficiency and the gene is CD36.
  • the disease comprises immunodeficiency with hyper-IgM type 3 and the gene is CD40.
  • the disease comprises hemolytic uremic syndrome and the gene is CD46. In some embodiments, the disease comprises complement hyperactivation, angiopathic thrombosis, or protein-losing enteropathy and the gene is CD55. In some embodiments, the disease comprises hemolytic anemia and the gene is CD59. In some embodiments, the disease comprises calcification of joints and arteries and the gene is CD73. In some embodiments, the disease comprises immunoglobulin alpha deficiency and the gene is CD79A. In some embodiments, the disease comprises C syndrome and the gene is CD96. In some embodiments, the disease comprises histiocytic sarcoma and the gene is CD163.
  • the disease comprises autosomal dominant deafness and the gene is CD 164. In some embodiments, the disease comprises immunodeficiency 25 and the gene is CD247. In some embodiments, the disease comprises methymalonic acidemia due to transcobalamin receptor defect and the gene is CD320.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, wherein the target sequence comprises a nucleotide sequence within a human safe harbor locus
  • the donor nucleic acid encodes a transgene that comprises a functional human protein that is expressed upon incorporation into the human safe harbor locus.
  • Embodiment 2 The composition of embodiment 1, wherein the effector protein comprises an amino acid sequence with at least 95% sequence identity to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • Embodiment 3 The composition of embodiment 1 or 2, wherein the effector protein comprises one or more amino acid substitutions relative to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • Embodiment 5 The composition 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 composition of embodiment 5, wherein the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • Embodiment 7 The composition of any one of embodiments 1-6, wherein the target sequence is in proximity to or adjacent to a protospacer adjacent motif (PAM) sequence comprising any one of the amino acid sequences recited in TABLE 3.
  • PAM protospacer adjacent motif
  • Embodiment 8 The composition of any one of embodiments 1-7, wherein the effector protein comprises a nuclear localization signal.
  • Embodiment 9 The composition of any one of embodiments 1-8, comprising a fusion partner protein linked to the effector protein.
  • Embodiment 10 The composition of embodiment 9, wherein the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via an amide bond.
  • Embodiment 11 The composition of any one of embodiments 1-10, wherein the nuclear localization signal comprises an amino acid sequence comprising any one of the amino acid sequences recited in TABLE 2.
  • Embodiment 13 The composition of any one of embodiments 1-11, wherein the human safe harbor locus is a gene or portion thereof that is expressed in the liver.
  • Embodiment 14 The composition of any one of embodiments 1-13, wherein the human safe harbor locus comprises any one of the nucleotide sequences recited TABLE 10.
  • Embodiment 15 The composition of any one of embodiments 1-13, wherein the human safe harbor locus is located in human chromosome 1, human chromosome 3, human chromosome 4, human chromosome 6, human chromosome 10, human chromosome 11, human chromosome 12, human chromosome 14, human chromosome 17, human chromosome 18, or human chromosome 19.
  • Embodiment 16 The composition of any one of embodiments 1-15, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4, TABLE 5, TABLE 6, TABLE 7, TABLE 9, and any combination thereof.
  • Embodiment 17 The composition of any one of embodiments 1-16, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4 and TABLE 5.
  • Embodiment 18 The composition of any one of embodiments 1-17, wherein the guide nucleic acid does not comprise a tracrRNA.
  • Embodiment 19 The composition of any one of embodiments 1-18, wherein the target sequence is a human gene.
  • Embodiment 20 The composition of any one of embodiments 1-19, wherein the target sequence comprises a nucleotide sequence with at least 90% sequence identity to any one of nucleotide sequences recited in TABLE 10.
  • Embodiment 22 The composition of any one of embodiments 1-21, wherein the functional human protein is select from any one of CFTR, DMD, GAA, Al AT, FXN, F8, F9, SOD1, C9, HTT, MECP2, SMN1, TARDBP, FUS, RHO, and USH2A or a functional variant or fragment thereof.
  • Embodiment 23 The composition of any one of embodiments 1-22, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266.
  • Embodiment 25 The composition of embodiment 24, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of SEQ ID NO: 348, 558, 401, 409, 473 and 486, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6 and 228-230.
  • Embodiment 26 The composition of embodiment 24, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of SEQ ID NO: 592, 600 and 602, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231.
  • Embodiment 27 The composition of embodiment 24, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 9
  • Embodiment 28 The composition of embodiment 23, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 652, 685, 705, 713, 777 and 790, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6 and 228-230.
  • Embodiment 29 The composition of embodiment 27, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 856, 864 and 866, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231.
  • Embodiment 32 A composition comprising: i. an effector protein, or a nucleic acid encoding the effector protein; ii. a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and iii.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • the guide nucleic acid is a crRNA, wherein the crRNA comprises a spacer sequence and a repeat sequence, wherein the spacer sequence hybridizes to a target sequence in a target nucleic acid, wherein the target sequence comprises a nucleotide sequence within a human safe harbor locus, wherein the target sequence comprises any one of the nucleotide sequences recited in TABLE 10
  • the donor nucleic acid encodes a functional human protein that upon expression after introduction in a human subject expression replaces the function of a corresponding nonworking or missing gene in the human subject.
  • Embodiment 33 The composition of embodiment 32, wherein the effector protein comprises one or more amino acid substitutions relative to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • Embodiment 36 The composition of embodiment 35, wherein the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • Embodiment 37 The composition of any one of embodiments 32-36, wherein the functional human protein is selected from any one of Al AT, CFTR, DMD, FXN, F8, F9, GAA, SOD1, C9, HTT, MECP2, SMN1, TARDBP, FUS, RHO, and USH2A or a functional variant or fragment thereof.
  • Embodiment 40 The composition of any one of embodiments 36-38, wherein the repeat sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 6.
  • Embodiment 41 The composition of any one of embodiments 36-39, wherein the repeat sequence is identical to any one of SEQ ID NO: 252-258, 1789, and 1848.
  • Embodiment 42 The composition of any one of embodiments 36-40, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266.
  • Embodiment 44 The composition of embodiment 42 or 43, wherein the spacer sequence is at least 90% identical to any one of SEQ ID NO: 301-510 and 536-575.
  • Embodiment 46 The composition of embodiment 45, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 605-839.
  • Embodiment 47 The composition of embodiment 46, wherein the crRNA comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 652, 685, 705, 713, 777 and 790, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6 and 228-230.
  • Embodiment 48 The composition of any one of embodiments 32-47, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NO: 6 and 228-230, and wherein the effector protein comprises one or more amino acid substitutions independently selected from E109R, H208R, K184R, K38R, L182R, Q183R, S 108R, S 198R, T114R or a combination thereof.
  • Embodiment 49 A composition comprising: i. an effector protein, or a nucleic acid encoding the effector protein; ii. a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and iii.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • the guide nucleic acid is a single guide RNA (sgRNA), wherein the sgRNA comprises a spacer sequence and a handle sequence, wherein the spacer sequence hybridizes to a target sequence in a target nucleic acid, wherein the handle sequence comprises one or more of an intermediary sequence, a repeat sequence, and a linker, wherein the target sequence comprises a nucleotide sequence within a human safe harbor locus, wherein the target sequence comprises any one of the nucleotide sequences recited in
  • the donor nucleic acid encodes a functional human protein that upon expression after introduction in a human subject expression replaces the function of a corresponding nonworking or missing gene in the human subject.
  • Embodiment 52 The composition of embodiment 50 or 51, wherein the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues.
  • Embodiment 53 The composition of embodiment 52, wherein the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • Embodiment 55 The composition of any one of embodiments 49-54, wherein the repeat sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 6.
  • Embodiment 56 The composition of any one of embodiments 49-55, wherein the repeat sequence is identical to any one of SEQ ID NO: 252-258, 1789 and 1848.
  • Embodiment 57 The composition of any one of embodiments 49-56, wherein the handle is at least 90% identical to any one of the nucleotide sequences recited in TABLE 7.
  • Embodiment 58 The composition of any one of embodiments 49-57, wherein the handle sequence is identical in a range of from 90% to 100% to any one of SEQ ID NO: 259-264.
  • Embodiment 60 The composition of any one of embodiments 49-59, wherein the linker sequence comprises a nucleotide sequence of SEQ ID NO: 265.
  • Embodiment 61 The composition of any one of embodiments 49-60, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266.
  • Embodiment 62 The composition of any one of embodiments 49-61, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 13 and 231.
  • Embodiment 63 The composition of any one of embodiments 49-62, wherein the spacer sequence is at least 90% identical to any one of SEQ ID NO: 536-604.
  • Embodiment 65 The composition of any one of embodiments 49-63, wherein the sgRNA comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 840-908.
  • Embodiment 66 The composition of embodiment 65, wherein the sgRNA comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NO: 856, 864 and 866, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231.
  • Embodiment 67 The composition of any one of embodiments 49-66, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231, 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.
  • Embodiment 68 The composition of any one of embodiments 49-66, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 231, and 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.
  • Embodiment 69 A composition comprising: i. an effector protein, or a nucleic acid encoding the effector protein; ii. a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and iii.
  • the effector protein comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences recited in SEQ ID NO: 47
  • the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, wherein the target sequence comprises a nucleotide sequence within intron 1 of a mammalian albumin gene
  • the donor nucleic acid encodes a functional human protein that upon expression after introduction in a human subject expression replaces the function of a corresponding nonworking or missing gene in the human subject.
  • Embodiment 70 The composition of embodiment 69, wherein the effector protein comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47
  • Embodiment 71 The composition of embodiment 69 or 70, 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 72 The composition of any one of embodiments 69-71, wherein the effector protein comprises a nuclear localization signal.
  • Embodiment 73 The composition of embodiment 72, wherein the nuclear localization signal comprises an amino acid sequence that is identical to an amino acid sequence of TABLE 2.
  • Embodiment 74 The composition of any one of embodiments 69-73, further comprising a fusion partner protein linked to the effector protein.
  • Embodiment 75 The composition of embodiment 74, wherein the fusion partner protein is directly fused to the N terminus or C terminus of the effector protein via an amide bond.
  • Embodiment 76 The composition of any one of embodiments 69-75, wherein the spacer sequence comprises a nucleotide sequence that has at least 90% sequence identity to any one of nucleotide sequences recited in TABLE 4.
  • Embodiment 78 The composition of any one of embodiments 69-77, wherein the guide nucleic acid comprises a repeat sequence that is at least 90% identical to SEQ ID NO: 254.
  • Embodiment 79 The composition of any one of embodiments 69-78, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of the guide RNA sequences recited in TABLE 9.
  • Embodiment 80 The composition of any one of embodiments 69-79, wherein the guide nucleic acid does not comprise a tracrRNA.
  • Embodiment 81 The composition of any one of embodiments 69-80, wherein the guide nucleic acid is a crRNA.
  • Embodiment 82 The composition of any one of embodiments 69-81, wherein the target sequence is a human gene.
  • Embodiment 83 The composition of any one of embodiments 69-82, wherein the target sequence comprises at least 17 contiguous nucleotides of any one of the nucleotide sequences recited in TABLE 10 or a reverse complement thereof.
  • Embodiment 84 The composition of any one of embodiments 69-83, wherein the composition cleaves both strands of the human albumin gene.
  • Embodiment 85 The composition of any one of embodiments 69-84, wherein the donor nucleic acid comprises at least 90% sequence identity to any one of the nucleotide sequences recited in TABLE 11.
  • Embodiment 86 A composition comprising: an effector protein, or a nucleic acid encoding the effector protein; a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid; and a donor nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 47, wherein the guide nucleic acid is a crRNA, wherein the crRNA comprises a spacer sequence and a repeat sequence, wherein the spacer sequence hybridizes to a target sequence in a target nucleic acid, wherein the spacer sequence is identical to any one of SEQ ID NO: 551-575, wherein the repeat sequence is identical to SEQ ID NO: 254, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266, and wherein the donor nucleic acid encodes a functional human protein that upon expression after introduction in a human subject expression replaces the function of a corresponding nonworking or missing gene in the human subject.
  • Embodiment 87 The composition of embodiment 86, wherein the guide nucleic acid is at least 90% identical to any one of SEQ ID NO: 815-839.
  • Embodiment 89 The composition of any one of embodiments 1-87, comprising a nucleic acid expression vector, wherein the expression vector comprises at least one of the nucleic acid encoding the effector protein; the nucleic acid encoding the guide nucleic acid; and the donor nucleic acid.
  • Embodiment 90 The composition of embodiment 89, wherein the nucleic acid expression vector is a viral vector.
  • Embodiment 91 The composition of embodiment 90, wherein the viral vector is an adeno associated viral (AAV) vector.
  • Embodiment 92 The composition of any one of embodiments 1-91, wherein the nucleic acid encoding the effector protein is a messenger RNA.
  • Embodiment 94 A pharmaceutical composition, comprising the composition of any one of embodiments 1-93; and a pharmaceutically acceptable excipient.
  • Embodiment 95 A system comprising components for introduction of a donor nucleic acid encoding a functional human protein into a human safe harbor locus, wherein the components comprise: an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence having at least 90% sequence identity to any one of amino acid sequences recited in TABLE 1A and TABLE IB; a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, and wherein the target sequence comprises a nucleotide sequence within the human safe harbor locus; and the donor nucleic acid.
  • Embodiment 96 The system of embodiment 95, wherein the effector protein comprises one or more amino acid substitutions relative to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • Embodiment 97 The system of embodiment 96, wherein the one or more amino acid substitutions independently comprise one or more conservative substitutions, one or more nonconservative substitutions, or combinations thereof.
  • Embodiment 98 The system of embodiment 96 or 97, wherein the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues.
  • Embodiment 99 The system of embodiment 98, wherein the positively charged amino acid residue is independently selected from Lys (K), Arg (R), and His (H).
  • Embodiment 100 The system of any one of embodiments 95-99, wherein the target sequence is in proximity to or adjacent to a protospacer adjacent motif (PAM) sequence comprising any one of the nucleotide sequences recited in TABLE 3, and wherein the effector protein and guide form a complex that modifies the human safe harbor locus upon hybridization of the guide nucleic acid to the target sequence and recognition of the PAM sequence by the effector protein.
  • PAM protospacer adjacent motif
  • Embodiment 101 The system of any one of embodiments 95-100, wherein the target sequence comprises any one of the nucleotide sequences recited in TABLE 10.
  • Embodiment 102 The system of embodiment 101, wherein the spacer sequence is at least 90% identical to any one of nucleotide sequences recited in TABLE 4 and TABLE 5, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, 13, 47 and 228-231.
  • Embodiment 103 The system of any one of embodiment 95-102, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266.
  • Embodiment 104 The system of embodiment 103, wherein the spacer sequence is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4.
  • Embodiment 105 The system of embodiment 104, wherein the guide nucleic acid comprises a nucleotide sequence that is at least 90% identical to any one of the nucleotide sequences recited in TABLE 9
  • Embodiment 106 A method of expressing a functional human protein in a cell, the method comprising contacting the cell with the composition of any one of embodiments 1-93, or the system of any one of embodiments 95-105.
  • Embodiment 107 A method of treating a disease associated with a mutation or aberrant expression of a human protein in a subject in need thereof, the method comprising administering to the subject the composition of any one of embodiments 1-93.
  • Embodiment 108 The method of embodiment 107, wherein the subject has a genetic disorder.
  • Embodiment 109 The method of embodiment 108, wherein the genetic disorder is a monogenic disorder.
  • Embodiment 110 The method of any one of embodiments 107-109, wherein the subject has cystic fibrosis, muscular dystrophy, Freidreich’s ataxia, amylotrophic lateral scleorsis, hemophilia, Huntington’s disease, retinal dystrophy, Rett syndrome, sickle cell disease, or a combination thereof.
  • Embodiment 111 The method of any one of embodiments 107-110, wherein the subject has a reduced activity of the human protein prior to the administering.
  • Embodiment 112. The method of any one of embodiments 107-110, wherein the subject has no activity of the human protein prior to the administering.
  • Embodiment 113 The method of any one of embodiments 107-112, wherein the subject has one or more genetic mutations.
  • Embodiment 114 The method of embodiment 113, 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.
  • SNP single nucleotide polymorphism
  • Embodiment 115 The method of any one of embodiments 107-114, wherein the mutation is associated with one or more of protein expression, protein activity, and protein stability.
  • Embodiment 116 The method of embodiment 115, wherein the mutation results in reduced expression of the human protein, reduced activity of human protein, reduced half-life of the human protein, or combinations thereof, in the cell relative to a cell without the mutation, before the treatment.
  • Embodiment 117 A system comprising components for introduction of a donor nucleic acid into intron 1 of a human albumin gene, wherein the components 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 90% identical to the amino acid sequence recited in SEQ ID NO: 47; a guide nucleic acid, or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a target sequence in a target nucleic acid, and wherein the target sequence comprises a nucleotide sequence within intron 1 of albumin gene; and the donor nucleic acid, wherein the donor nucleic acid encodes a functional human protein that upon expression after introduction in a human subject expression replaces the function of a corresponding nonworking or missing gene in the human subject.
  • Embodiment 118 The system of embodiment 117, wherein the guide nucleic acid comprises at least one nucleotide sequence selected from any one of TABLES 3, 4, and 5.
  • Embodiment 120 A method of treating a disease associated with a mutation or aberrant expression of a protein in a subject in need thereof, the method comprising administering to the subject the composition of any one of embodiments 69-93.
  • Embodiment 121 The method of embodiment 120, wherein the subject has a genetic disorder.
  • Embodiment 122 The method of embodiment 120 or 121, wherein the mutation comprises a point mutation, a single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof.
  • SNP single nucleotide polymorphism
  • Embodiment 123 The method of any one of embodiments 170-116 or 119-122, wherein the method is performed in a cell.
  • Embodiment 124 The method of any one of embodiments 170-117 or 119-122, wherein the method is performed in vivo.
  • Embodiment 125 A cell comprising the composition of any one of embodiments 1-93 or 69- 87.
  • Embodiment 126 A cell that comprises the target nucleic acid modified by the composition of any one of embodiments 1-93 or 69-87.
  • Embodiment 128 The cell of any one of embodiments 125-127, wherein the cell is a mammalian cell.
  • Embodiment 129 The cell of any one of embodiments 125-128, wherein the cell is a human cell.
  • Embodiment 130 The cell of any one of embodiments 125-129, wherein 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
  • Embodiment 131 A population of cells that comprises at least one cell of any one of embodiments 125-130.
  • Embodiment 133 The system of embodiment 132, wherein the effector protein comprises an amino acid sequence with at least 95% sequence identity to any one of the amino acid sequences recited in TABLE 1A and TABLE IB
  • Embodiment 135. The system of embodiment 134, wherein the one or more amino acid substitutions independently comprise one or more conservative substitutions, one or more nonconservative substitutions, or combinations thereof.
  • Embodiment 136 The system of embodiment 134 or 135, wherein the one or more amino acid substitutions comprise one or more substitutions with a positively charged amino acid residues.
  • Embodiment 139 The system of any one of embodiments 132-138, wherein the effector protein comprises a nuclear localization signal.
  • Embodiment 140 The system of any one of embodiments 132-139, comprising a fusion partner protein linked to the effector protein.
  • Embodiment 145 The system of any one of embodiments 132-144, wherein the guide nucleic acid does not comprise a tracrRNA.
  • Embodiment 146 The system of any one of embodiments 132-145, wherein the system is capable of cleaving both strands of the human safe harbor locus prior to incorporation of the donor nucleic acid.
  • Embodiment 147 The system of any one of embodiment 132-146, wherein the functional human protein is select from any one of CFTR, DMD, GAA, Al AT, FXN, F8, F9, SOD1, C9, HTT, MECP2, SMN1, TARDBP, FUS, RHO, and USH2A or a functional variant or fragment thereof.
  • Embodiment 148 The system of any one of embodiments 132-147, wherein the target sequence comprises a nucleotide sequence within SEQ ID NO: 266.
  • Embodiment 149 The system of embodiment 148, wherein the spacer sequence is at least 90% identical to any one of the nucleotide sequences recited in TABLE 4, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, 13, 47, and 228-231.
  • Embodiment 150 The system of embodiment 149, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of SEQ ID NO: 348, 558, 401, 409, 473 and 486, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 6, and 228-230.
  • Embodiment 151 The system of embodiment 149, wherein the guide nucleic acid comprises a spacer sequence that is at least 90% identical to any one of SEQ ID NO: 592, 600 and 602, and wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

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Abstract

La présente divulgation concerne des compositions, des systèmes et des procédés comprenant des protéines effectrices et des utilisations associées. Ces protéines effectrices peuvent être caractérisées en tant que protéines associées à CRISPR (Cas). Diverses compositions, divers systèmes et procédés de la présente divulgation peuvent tirer profit des activités de ces protéines effectrices pour la modification et l'ingénierie d'acides nucléiques.
PCT/US2023/066848 2022-05-10 2023-05-10 Compositions de protéines effectrices et procédés d'utilisation associés WO2023220654A2 (fr)

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