WO2025106934A1 - Enzymes à domaines ruvc - Google Patents

Enzymes à domaines ruvc Download PDF

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Publication number
WO2025106934A1
WO2025106934A1 PCT/US2024/056299 US2024056299W WO2025106934A1 WO 2025106934 A1 WO2025106934 A1 WO 2025106934A1 US 2024056299 W US2024056299 W US 2024056299W WO 2025106934 A1 WO2025106934 A1 WO 2025106934A1
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sequence
seq
nos
identity
engineered
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Inventor
Brian C. Thomas
Lisa ALEXANDER
Christopher Brown
Daniela S.A. Goltsman
Rebecca LAMOTHE
Sarah Laperriere
Isabel NOCEDAL
Kartik RALLAPALLI
Nicole Thomas
Leopold D'espaux
Molly BROTHERS
Shweta BELUR
Margaret HACKNEY
Thao LUONG
Junkai WANG
Bryan ANGGITO
Dinh Le
Brandon PEKAREK
Kate SENGER
Paul Szymanski
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Metagenomi Therapeutics Inc
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Metagenomi Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

  • engineered nuclease systems comprising: an endonuclease comprising a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 47-52, 659-698, 983-1010, 1263-1282, 1158-1159, and 975-981; and an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.
  • the endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 47-52, 659-698, 983-1010, 1263-1282, 1158-1159, and 975-981.
  • the endonuclease comprises a sequence having 90% sequence identity to any one of SEQ ID NOs: 47-52, 659-698, 983-1010, 1263-1282, 1158-1159, and 975- 981.
  • the engineered guide polynucleotide comprises a crRNA and a tracrRNA.
  • the tracrRNA comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 710-722, 726-744, 745-767, 699, 700-702, and 703-709.
  • the tracrRNA comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 710-722, 726-744, 745-767, 699, 700-702, and 703-709.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA.
  • the endonuclease is not a Cas9 endonuclease.
  • the endonuclease has less than 80% identity to a Cas9 endonuclease.
  • the endonuclease binds non-covalently to the engineered guide polynucleotide. [0014] In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47 ; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1137-1144, 1283- 1392, 1502-1509, and 1786-2045.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 48; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to SEQ ID NOs: 1153-1156, 1393-1493, 1494- 1501, and 1510-1525.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 57-58, 77-88, 101-119, 139-150, 163- 181, 201-212, 225-243, 343-374, 407-413, 421-433, 447-453, 461-472, 485-491, 499-511, 525- 531, 539-551, 565-578, 593-625, 710-722, 927-942, 945-950, 9
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 51 or SEQ ID NO: 1264; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 61.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NOs: 659, 660, 1158, 1159, and 1267-1277; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 726-744, 843-880, 1011-1055, and 1145-1152.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 661-678 and 1278-1282; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 745-767 and 881-926.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 659; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 699 and 973-974.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 696-698; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 700-702 and 957-960.
  • engineered nuclease systems comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 975-981; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 703-709, 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide is a single guide nucleic acid.
  • the engineered guide polynucleotide is a dual guide nucleic acid.
  • the engineered guide polynucleotide is RNA.
  • the endonuclease is not a Cas9 endonuclease.
  • the endonuclease binds non-covalently to the engineered guide polynucleotide.
  • the endonuclease is covalently linked to the engineered guide polynucleotide. [0031] In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.
  • Described herein, in certain embodiments, are methods for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system discloses herein.
  • modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.
  • the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.
  • the modification is in vitro.
  • the modification is in vivo.
  • the modification is ex vivo.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1161-1262, 1526-1785, and 2046-2050.
  • Described herein, in certain embodiments, are methods of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease systems disclosed herein.
  • the method further comprises selecting cells comprising the modification.
  • HAO1 hydroxy acid oxidase 1
  • methods of modifying a hydroxy acid oxidase 1 (HAO1) gene comprising contacting the HAO1 gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • ATP7B ATPase copper transporting beta
  • an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1056-1081.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1206-1231.
  • adeno-associated virus integration site 1 comprising contacting AAVS1 using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1010, 1263, 47, 659, 660, 1158, 1159, and 1267-1277; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 710-722.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 21-433, 461-472, 499- 511, 539-551, 1082, 1098-1102, 1393-1493, 1020-1049, and 1051-1055.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 59-60, 120-138, 182-200, 243-262, 375-406, 434-446, 473-484, 512-524, 552-564, 1170-1205, 1232, and 1248-1252.
  • Described herein, in certain embodiments, are methods of modifying T cell receptor alpha constant (TRAC) comprising contacting TRAC using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-10101263, 48, 659, 660, 1158, 1159, and 1267-1277; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 710-722.
  • TRAC T cell receptor alpha constant
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 61, 77-88, 139-150, 201-212, 407-413, 447-453, 485-491 525-531, 1096-1097, 1011-1019, 1050, 1494-1501, and 1510-1525.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 62, 89-100, 151-162, 213-224, 414-420, 454-460, 492-489, 532-538, 1161- 1168, and 1200.
  • Described herein, in certain embodiments, are methods of modifying an albumin gene comprising contacting the albumin gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 49- 51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.
  • the engineered guide polynucleotide comprises a sequence having any SEQ ID NO: 1083-1094.
  • the target nucleic acid sequence comprises a sequence having SEQ ID NO: 1233-1244.
  • Described herein, in certain embodiments, are methods of modifying a beta-2- microglobulin (B2M) gene comprising contacting the B2M gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 710-722.
  • an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 565-578 and 1095.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 579-592 and 1245.
  • Described herein, in certain embodiments, are methods of modifying a hemoglobulin subunit beta (HBB) gene comprising contacting the HBB gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 710-722.
  • HBB hemoglobulin subunit beta
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 593-625.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 626-658.
  • Described herein, in certain embodiments, are methods of modifying a phenylalanine hydroxylase (PAH) gene comprising contacting the PAH gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1113-1122.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1253-1262.
  • Described herein, in certain embodiments, are methods of modifying an ataxin 2 (ATXN2) gene comprising contacting the ATXN2 gene using an engineered nuclease system comprising an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.
  • ATXN2 ataxin 2
  • the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1786-2045, and 2051-2055.
  • the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1526-1785, and 2046-2050.
  • cells comprising the engineered nuclease systems disclosed herein.
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.
  • the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.
  • the cell is an engineered cell.
  • the cell is a stable cell.
  • FIG. 1 depicts the gene editing outcomes at the DNA level for TRAC (A1-D2) and AAVS1 (E2-G4) in K562 cells as described in Example 4.
  • FIG. 2 depicts the gene editing outcomes at the DNA level for TRAC in K562 cells as described in Example 5.
  • FIG. 3A depicts analysis of the gene editing outcomes of first engineering round of the MG71-2 scaffold with guide hAAVSl C3 at the DNA level.
  • FIG. 3B depicts analysis at the DNA level of second round of engineering for MG71-2 scaffold with guide hAAVSl C3.
  • FIG. 4 depicts analysis of the gene editing outcomes with original vs optimized MG71- 2 scaffold at the DNA levels. Spacers indicated on the Y axis were tested with either a 22nt or 24nt spacer and either the original or shortened MG71-2 scaffold 11.
  • FIGs. 5A-5B depict analysis of the gene editing outcomes with original vs optimized MG71-2 scaffold at the DNA levels.
  • FIG. 5A depicts editing outcomes with MG71-2 engineered guides (scaffold 21) at exon 2 of the human B2M gene.
  • FIG. 5B depicts editing outcomes with MG71-2 engineered guides (scaffold 21) at exon 3 of the human HBB gene.
  • FIG. 6 depicts a phylogenetic tree of the reconstructed ancestral variants of the MG71 family based on multiple sequence alignment. Ancestral sequences were generated for nodes of interest (shown with black circles).
  • FIG. 7 depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG71-28 obtained from the in vitro cleavage assay and NGS sequencing.
  • the MG71 family display a range of PAM specificities, with preference for an A in the 4th position.
  • FIG. 8 depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG73-6 obtained from the in vitro cleavage assay and NGS sequencing.
  • the nuclease MG73-6 was active in vitro with an nnRNCTW PAM.
  • FIG. 9 depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG74-8 obtained from the in vitro cleavage assay and NGS sequencing.
  • the MG74 family has A/C rich PAMs.
  • FIG. 10 depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG87-102 obtained from the in vitro cleavage assay and NGS sequencing.
  • the MG87 family nucleases have the strongest preference in the 5th and 6th bases from the spacer.
  • FIG. 11 depicts a SeqLogo of the protospacer adjacent motif (PAM) recognized by MG88-11 obtained from the in vitro cleavage assay and NGS sequencing.
  • the MG88 family tend to have purine-rich PAMs in the second through fourth positions.
  • FIG. 12A-12B depict SeqLogos of the protospacer adjacent motifs (PAMs) recognized by MG71 (FIG. 12A: MG71-42; FIG. 12B: MG71-43) ancestors obtained from the in vitro cleavage assay and NGS sequencing.
  • the MG71 ASR proteins are active and have similar or more relaxed PAMs as the metagenomic protein MG71-2.
  • FIG. 13 depicts the gene editing outcomes at the DNA level (shown in % indel) for AAVS1 in K562 cells.
  • FIG. 14 depicts a phylogenetic tree of the reconstructed ancestral variants of the MG71 family.
  • the phylogenetic tree was inferred with FastTree from a MAFFT multiple sequence alignment. Ancestral sequences were generated for nodes of interest (shown with white circles).
  • FIG. 15 depicts alphafold2 predicted structure of MG71-2 with the gRNA and R-loop from crystal structure of SpCas9 bound to substrate DNA and guide (PDB ID: 4UN3).
  • FIG. 15 shows predicted structure of MG71-2 after PID mutations. Residues predicted to be within the first interaction shell (5A) of the PAM sequence are depicted as sticks and labelled.
  • FIG. 16 depicts in vitro PAMs of MG71-2 and MG71-43 single point mutants.
  • the nucleases were expressed in an in vitro transcription/translation reaction, then incubated with guide a 5N plasmid library. Cut plasmids were sequenced by NGS and aligned to generate the PAM.
  • the first nucleotide “T” in the 0 position is masked as it represents the constant flanking base adjacent to the spacer in the 5N PAM enrichment library (TNNNNN).
  • FIG. 17 depicts in vitro PAMs of MG71 ancestral reconstructions.
  • the nucleases were expressed in an in vitro transcription/translation reaction, then incubated with guide a 5N plasmid library. Cut plasmids were sequenced by NGS and aligned to generate the PAM.
  • the first nucleotide “T” in the 0 position is masked as it represents the constant flanking base adjacent to the spacer in the 5N PAM enrichment library (TNNNNN).
  • FIG. 18 depicts MG87-70 tested for gene editing activity in human cells (K562 cells) with two doses of guide via nucleofection. 72 hrs later cells were harvested and gDNA was prepped to evaluate editing via NGS.
  • FIG. 19 depicts MG87-21 tested for gene editing activity in human cells (K562 cells) with 650 pmol of gRNA and 500 ng of mRNA per well via nucleofection. 72 hrs later cells were harvested and gDNA was prepped to evaluate editing via NGS.
  • FIG. 20 depicts MG71-2 and MG71-43 at ATP7B in PHH cells screened for active guides to the intronic region of ATP7B, between exon 5 and exon 6. Of 94 guides screened, 24 guides were identified as active >1% in PHH cells via NGS.
  • FIG. 21 depicts MG71-2 and MG71-43at ATP7B in Hep3B cells screened for active guides to the intronic region of ATP7B, between exon 5 and exon 6. Of 94 guides screened, 26 guides were identified as active >1% in Hep3B cells via NGS.
  • FIG. 22 depicts MG71-2 and MG71-43 at Albumin in PHH cells screened for active guides to the intron 1 region of albumin. Of 19 guides screened, 10 guides were identified as active > 1% in PHH cells for one or both nucleases. Additionally, 11 guides were designed only for MG71-43; of these guides, 2 were active in PHH.
  • FIGs. 23A-23C depict specificity of MG71-2 and MG71-43 showing MG71 family displaying high specificity in mammalian cells.
  • Cells were assessed for double strand break (DSB) formation by co-nucleofection mRNA, gRNA, and annealed dsODN.
  • the dsODN could be incorporated into DSBs and used as a priming site for NGS library prep, allowing for unbiased sampling of all DSBs created in the cell.
  • the percent of dsODN reads coming from the desired target site was mapped for up to three replicates for nuclease MG71-2 (FIG. 23A). For all 8 guides, all the reads passing analysis criteria come from the on-target site.
  • FIG. 23B discloses SEQ ID NOs: 2160-2163, 2160-2163 and 2160-2163, respectively, in order of appearance.
  • FIGs. 24A-24B depict specificity of MG87-70 displaying high specificity in mammalian cells.
  • Cells were assessed for double strand break (DSB) formation by co- nucleofection mRNA, gRNA, and annealed dsODN.
  • the dsODN could be incorporated into DSBs and used as a priming site for NGS library prep, allowing for unbiased sampling of all DSBs created in the cell.
  • the percent of dsODN reads coming from the desired target site was mapped for up to 3 replicates for nuclease MG87-70. For 4/7 guides, all the reads passing analysis criteria come from the on-target site (FIG. 24A).
  • FIG. 24B discloses SEQ ID NOs: 2164-2167, 2164-2167 and 2164-2167, respectively, in order of appearance.
  • FIG. 25 depicts pooled screening of MG71 chimeras at PAH p.R408W in engineered immortalized K562 cells. Each nuclease was delivered as codon-optimized mRNA along with 10 guide RNAs that target the SNV of interest.
  • FIG. 26 depicts human HAO1 MG21-1 engineered guide RNA activity.
  • FIG. 27 depicts human AAVS1 MG23-1 engineered guide RNA activity.
  • FIG. 28 depicts effect of spacer length on gRNA activity (% indel). Analysis of geneediting outcomes at the DNA level for guides of different spacer lengths on 1-2 different chemically modified backbones from three different Type II editing systems (MG23-1, MG21-1, and MG71-2).
  • hAAVSl-23-l-Gl-l (SEQ ID NOs: 1486-1493), hTRAC-23-l-H7-l (SEQ ID NOs: 1494-1500), hHA01-21-l-Fl-78 (SEQ ID NOs: 1502-1505), hHA01-21-l-Fl-157 (SEQ ID NOs: 1506-1509), hTRAC-71-2-C2-12 (SEQ ID NOs: 1510-1517), hTRAC-71-2-C2-255 (SEQ ID NOs: 1518-1525).
  • FIG. 29 depicts guide RNA targeting the human Atxn2 gene with MG21-1 in K562 cells.
  • FIG. 29 further shows analysis of Atxn2 guides with MG21-1 mRNA in K562 cells. Nucleofection of MG21-1 along with the matching A txn2 guide RNA (500 ng mRNA/150 pmol guide) was performed into K562 (100,000 cells). Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing. Graphs illustrate guides tested, targeting all exons of Atxn2 region of interest.
  • FIG. 30 depicts validation of a subset of MG21-1 synthetic guides on the Atxn2 gene in human neural progenitor cells.
  • FIG. 30 further shows analysis of Atxn2 guides with MG21-1 mRNA in human neural progenitor cells.
  • NPC cells were co-transfected with 200 ng of mRNA and chemically-synthesized sgRNA targeting Atxn2.
  • Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. The target genomic regions were amplified from the extracted gDNA and the amplicons were sequenced on an Illumina MiSeq platform. Sequences were analyzed using a proprietary Python script to quantify gene editing efficiency. % OOF denotes out of frame mutations.
  • FIG. 31 depicts mouse versions of active human synthetic guides showing Atxn2 gene editing activity in mouse neuro-2A cells.
  • the neuro-2a cells were transfected using a serial dilution of MG21-1 mRNA and mouse surrogate Atxn2 guides via nucleofection. The highest concentration in the serial dilution was 500 ng of mRNA and 150 pmol of guide, followed by a 1:3 serial dilution series.
  • Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. The target genomic regions were amplified from the extracted gDNA with primers designed for NGS-based sequencing. The amplicons were sequenced on an Illumina MiSeq platform and analyzed using a proprietary Python script to quantify gene editing efficiency.
  • FIG. 32 depicts design of AAV constructs bearing Atxn2 mouse surrogate guide RNAs.
  • the AAV cargo constructs were designed to express MG21-1 and a guide RNA from a single AAV.
  • the payload was flanked by two, 141 bp inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • a U6 polymerase III promoter was placed upstream of the guide RNA sequence in the forward orientation at the 5’ end of the cargo. This was followed by a CMV promoter driving the MG21- 1 open reading frame.
  • MG21-1 was N-terminally tagged with a V5 epitope.
  • a synthetic polyadenylation sequence (SpA) follows MG21-1.
  • FIG. 33 depicts AAV constructs bearing MG21-1 and guides targeting mouse Atxn2 show gene editing activity in mouse neuro-2A cells.
  • the neuro-2a cells were transfected using a serial dilution of AAV cargo plasmids via lipofection. The highest plasmid concentration in the dilution series was 2 ug, followed by a 1:2 serial dilution.
  • Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. The target genomic regions were amplified from the extracted gDNA with primers designed for NGS-based sequencing. PCR products were purified and the amplicons were sequenced. Sequences were analyzed to quantify gene editing efficiency.
  • FIG. 34 depicts AAV constructs bearing MG21-1 and guides targeting mouse Atxn2 show gene editing activity in mouse Neuro-2A cells.
  • the neuro-2a cells were transfected using a serial dilution of AAV cargo plasmids via lipofection. The highest plasmid concentration in the dilution series was 500 ng, followed by a 1:2 serial dilution.
  • Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. The target genomic regions were amplified from the extracted gDNA with primers designed for NGS-based sequencing. PCR products were purified and the amplicons were sequenced. Sequences were analyzed using a proprietary Python script to quantify gene editing efficiency.
  • FIG. 35 depicts AAV constructs bearing MG21-1 and guides targeting mouse Atxn2 showing protein knockdown in mouse neuro-2A cells.
  • the neuro-2a cells were transfected with each of five AAV plasmids (1 pg of DNA) containing different mouse surrogate A txn2 guides using Lipofectamine 2000. Three days after transfection, cells were lysed, and 10 pg was run on a 4-20% Tris-Glycine gel. Proteins were transferred to a nitrocellulose membrane, blocked with 5% milk, then probed with the antibodies against the V5 epitope and Atxn2. Blots were reprobed with an actin antibody as a reference control. [0110] FIG.
  • AAV constructs bearing MG21-1 and guides targeting mouse Atxn2 show full-length genome packaging into AAV9 by alkaline gel electrophoresis.
  • the AAVs were produced using a transient triple co-transfection method in HEK293 cells involving a plasmid encoding adenoviral helper proteins, a plasmid encoding AAV replication protein and AAV9 capsid protein, and a cargo plasmid encoding guide and MG21-1 flanked between two AAV2 ITRs. After transfection, the cells were incubated at 37 °C for 72 hrs prior to harvest. A crude viral lysate was created, which was then treated prior to purification via cesium chloride density gradient ultracentrifugation. The AAVs were then subjected to 3 rounds of dialysis prior to loading on an alkaline agarose gel to visualize the size and integrity of the vector genomes.
  • FIG. 37 depicts in vivo study design, experimental procedure, and sample preparation.
  • 6-8-week-old C57BL/6J mice were injected with AAV9 into both the olfactory bulb and Ml region of the motor cortex using a custom built stereotactic delivery system based on the RWD automated stereotaxic instrument.
  • Ml injections consisted of a bilateral infusion of 750 nL of AAV9 infused at a rate of 5 nL/second at coordinates +1.00 AP, +/-1.50 ML, -1.50 DV relative to Bregma.
  • Olfactory bulb injections consisted of a bilateral infusion of 250 nL of AAV9 infused at a rate of 5 nL/second and three target depths, coordinates +4.00 AP, +/- 0.70 ML, -2.5/-2.0/- 1.5 DV relative to Bregma.
  • a retention time of 5 minutes post injection was added to enhance absorption of the AAV into the tissue.
  • Animals were sutured and allowed to recover until transferred back to their home cages.
  • Two weeks post injection animals were euthanized via deep inhalation of isoflurane.
  • Whole brains were dissected fresh, the olfactory bulb and the Ml regions were further dissected and processed for downstread nucleic acid extraction. Genomic DNA and mRNA was extracted from fresh tissue using DNA and RNA isolation kits.
  • FIG. 38 depicts tissue titer measurements demonstrate MG21-1 AAVs effectively transduce mouse olfactory bulb and motor cortex.
  • 6-8-week-old C57BL/6J mice were injected with AAV9 prepared in-house into both the olfactory bulb and Ml region of the motor cortex using a custom built stereotactic delivery system.
  • Two weeks post injection animals were euthanized via deep inhalation of isoflurane.
  • Whole brains were dissected fresh, the olfactory bulb and the Ml regions were further dissected and processed for downstream nucleic acid extraction.
  • FIG. 39 depicts MG21-1 transcripts are detected in the olfactory bulb and motor cortex after AAV9 injection.
  • mice 6-8-week-old C57BL/6J mice were injected with AAV9 prepared in-house into both the olfactory bulb and Ml region of the motor cortex using a custom built stereotactic delivery system. Two weeks post injection, animals were euthanized via deep inhalation of isoflurane. Whole brains were dissected fresh, the olfactory bulb and the Ml regions were further dissected and processed for downstream nucleic acid extraction. mRNA was extracted from fresh tissue and then used to produce cDNA by reverse transcription. To quantify the expression of MG21-1, 5ng of cDNA mRNA equivalent was loaded into a ddPCR reaction containing custom primers and probes targeting the central region of the MG21-1 sequence.
  • This assay was additionally multiplexed with an assay targeting the Cycl gene. Data were graphed in GraphPad Prism 10. MG21-1 copies were normalized by Cycl reference copies to produce a normalized MG21-1 copies/Cycl copies value. All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • FIG. 40 depicts MG21-1 packaged into AAV9 effectively knocks down Atxn2 mRNA in the olfactory bulb and motor cortex.
  • 6-8-week-old C57BL/6J mice were injected with AAV9 prepared in-house into both the olfactory bulb and Ml region of the motor cortex using a custom built stereotactic delivery system.
  • Two weeks post injection animals were euthanized via deep inhalation of isoflurane.
  • Whole brains were dissected fresh, the olfactory bulb and the Ml regions were further dissected and processed for downstread nucleic acid extraction.
  • mRNA was extracted from fresh tissue and then used to produce cDNA by reverse transcription.
  • Atxn2 To quantify the expression of Atxn2, we loaded 5 ng of cDNA mRNA equivalent to a ddPCR reaction containing an Atxn2 premade expression quantification assay. This assay was additionally multiplexed with a premade assay targeting the Cycl gene. Data were graphed in GraphPad Prism 10. Atxn2 mRNA copies were normalized to Cycl reference copies to produce a normalized Atxn2 copies/Cycl copies value. All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • FIG. 41 depicts a graph of indels within the mouse Atxn2 gene are detectable within mouse olfactory bulb and motor cortex. All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • FIG. 42 depicts a graph of the quantification of Atxn2 protein expression in olfactory bulb samples injected with AAV9. All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • FIG. 43 depicts data from stereotactic injection of an AAV encoding MG21-1 targeting Atxn2 showing target protein knockdown by imaging. Scale bars represent 70 pm.
  • FIG. 44 depicts data from stereotactic injection of an AAV encoding MG21-1_P1F12 guide shows Atxn2 protein knockdown by imaging. Scale bars represent 70 pm.
  • FIG. 45 depicts a schematic overview of the workflow. Single mismatches across every position of spacer and PAM were generated (1MM Target) and used to create the dual-target library members. The library was ordered and cloned into a lentiviral plasmid to create the plasmid library from which the lentiviral library was produced. K562 cells were infected with the dual-target lentibrary and following antibiotic selection the cells were edited via nucleofection and the gDNA harvested. After NGS sequencing the data was analyzed.
  • FIGs. 46A and 46B depict bar graphs showing GC content and Percent Indels for library sgRNAs.
  • the range of GC contents (FIG. 46A) and percent indels (FIG. 46B) for the guides from which the library was designed are plotted by nuclease.
  • FIG. 47 depicts a schematic overview of the reference structure of dual-target oligo library member.
  • FIG. 48 depicts graphs showing editing controls.
  • Control oligonucleotides were generated to simulate four possible editing events: cleavage at the on-target, cleavage at the off- target, cleavage at both targets, and no cleavage.
  • These control dual-target sequences possessed identical on- and off-target protospacers, with cleavage events regulated by their active or inactive PAM sequences.
  • To generate inactive PAMs bases were substituted in the active PAM consensus sequence with alternatives that are not recognized by the Cas system. Editing at the left or right end of the dual-target controls, following nucleofection with guides across MG71-2 and MG21-1, was plotted.
  • FIG. 49 depicts the generation of a nuclease-specific single mismatch tolerance profile.
  • Raw indel percentages at mismatched targets and their corresponding on-target were calculated for each library member.
  • the raw indel percentages were normalized across each position by taking the ratio of observed off:on target editing, termed mismatch tolerance, for each guide.
  • a nuclease-specific single mismatch tolerance profile was generated by plotting the mean and 95% confidence intervals of these single mismatch tolerances, by position, of all guides for a given nuclease.
  • FIG. 50 depicts a graph showing single mismatch tolerance of 22 nucleotide spacers for MG71-2. 25 guides targeting human B2M, TRAC, AAVS1 loci were used to design dual-target library members in which the off-target sequence differs by 1 mismatch between the off and on- target sequence. The raw indel percentages at mismatched targets compared to on-target library members were normalized by taking the ratio of off:on target editing, termed mismatch tolerance, observed by position for each guide and a nuclease specific profile was generated by plotting the mean and 95% confidence intervals of single mismatch tolerance by position. [0125] FIG. 51 depicts a graph showing single mismatch tolerance of 24 nucleotide spacers for MG71-2.
  • FIG. 52 depicts a graph showing single mismatch tolerance of MG71-2 with 22 and 24 nucleotide spacers overlaid.
  • 25 guides of 22nt length targeting human B2M, TRAC, AAVS1 loci, and 7 guides of 24nt length targeting the human HBB locus were used to design dual-target library members in which the off-target sequence differs by 1 mismatch between the off and on- target sequence.
  • the raw indel percentages at mismatched targets compared to on-target library members were normalized by taking the ratio of off:on target editing, termed mismatch tolerance, observed by position for each guide and a nuclease specific profile was generated by plotting the mean and 95% confidence intervals of single mismatch tolerance by position. The two plots were overlaid to visualize.
  • FIG. 53 depicts a graph showing single mismatch tolerance of MG21-1.
  • 21 guides targeting human PDCD1, HA01, IL 17, and TRAC loci were used to design dual-target library members in which the off-target sequence differs by 1 mismatch between the off and on-target sequence.
  • the raw indel percentages at mismatched targets compared to on-target library members were normalized by taking the ratio of off:on target editing, termed mismatch tolerance, observed by position for each guide and a nuclease specific profile was generated by plotting the mean and 95% confidence intervals of single mismatch tolerance by position.
  • FIG. 54 depicts a graph showing MG71-2 PAM preference. PAM preference scores were calculated for MG71-2 by converting the mismatch tolerance scores and their respective off-target PAMs into information-based representation matrices using logomaker.
  • FIG. 55 depicts a graph showing MG21-1 PAM preference. PAM preference scores were calculated for MG21-1 by converting the mismatch tolerance scores and their respective off-target PAMs into information-based representation matrices using logomaker.
  • FIG. 56 depicts an MG71-2 protospacer base preference heatmap. Heatmaps were created to illustrate the protospacer base preference at each mismatch position for different nucleases. The heatmaps were generated by grouping the data points by nuclease, mismatch location, and mismatched protospacer base, and then calculating the mean off-on ratio and 95% confidence interval for each group.
  • FIG. 57 depicts an MG21-1 protospacer base preference heatmap. Heatmaps were created to illustrate the protospacer base preference at each mismatch position for different nucleases. The heatmaps were generated by grouping the data points by nuclease, mismatch location, and mismatched protospacer base, and then calculating the mean off-on ratio and 95% confidence interval for each group.
  • SEQ ID NO: 47 shows the full-length peptide sequence of an MG21 nuclease.
  • SEQ ID NOs: 1137-1144 show the nucleotide sequences of sgRNAs engineered to function with an MG21 nuclease.
  • SEQ ID NO: 48 shows the full-length peptide sequence of an MG23 nuclease.
  • SEQ ID NOs: 1153-1156 show the nucleotide sequences of sgRNAs engineered to function with an MG23 nuclease.
  • SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 show the full-length peptide sequences of MG71 nucleases.
  • SEQ ID NOs: 53-54 show the peptide sequences of PAM-interacting domains of MG71 nucleases.
  • nnRMYnn, nnnACTnn nNNRMT, nRNACT, nNNACT, nRNRCT, nNNRHY, nNNRMT, nNNRCY, nNNRYY, nNNRMT, nNNRCY, nNWRAT, nNNRMT, nNNRHT, nNNRHY, nNNRHY, nNNRHY, nRNRYY, nRNRCN, nNNRYY, nRNRNT, nNNAWT, nNRAMW, nNARMY, nNNRCT, nNNRYT, nNARYY, nNNRCY, and nNNRYY are PAM sequences compatible with MG71 nucleases.
  • SEQ ID NOs: 710-722 show the nucleotide sequences of MG71 tracrRNAs.
  • SEQ ID NOs: 779-791 show the nucleotide sequences of MG71 CRISPR repeats.
  • SEQ ID Nos: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434 show the nucleotide sequences of sgRNAs engineered to function with an MG71 nuclease.
  • “n” can be any nucleotide.
  • SEQ ID NOs: 51 and 1264 show the full-length peptide sequence of a MG73 nuclease.
  • SEQ ID NO: 55 shows the peptide sequence of a PAM-interacting domain of a MG73 nuclease.
  • nnRnTTnn is a PAM sequence compatible with a MG73 nuclease.
  • SEQ ID NO: 52 shows the full-length peptide sequence of a MG89 nuclease.
  • SEQ ID NO: 56 shows the peptide sequence of a PAM-interacting domain of a MG89 nuclease.
  • nnnnCC is a PAM sequence compatible with a MG89 nuclease.
  • SEQ ID Nos: 659-660, 1158-1159, and 1267-1277 show the full-length peptide sequences of MG87 nucleases.
  • SEQ ID NOs: 726-744 show the nucleotide sequences of MG87 tracrRNAs.
  • SEQ ID NOs: 795-813 show the nucleotide sequences of MG87 CRISPR repeats.
  • SEQ ID NOs: 843-880 and 1145-1152 show the nucleotide sequences of sgRNAs engineered to function with an MG87 nuclease.
  • SEQ ID NOs: 661-678 and 1278-1282 show the full-length peptide sequences of MG88 nucleases.
  • SEQ ID NOs: 745-767 show the nucleotide sequences of MG88 tracrRNAs.
  • SEQ ID NOs: 814-836 show the nucleotide sequences of MG88 CRISPR repeats.
  • SEQ ID NOs: 881-926 show the nucleotide sequences of sgRNAs engineered to function with an MG88 nuclease.
  • SEQ ID NO: 695 shows the full-length peptide sequence of a MG17 nuclease.
  • SEQ ID NO: 699 shows the nucleotide sequence of a MG17 tracrRNA.
  • SEQ ID NO: 768 shows the nucleotide sequence of a MG17 CRISPR repeat.
  • SEQ ID NOs: 973-974 show the nucleotide sequences of sgRNAs engineered to function with an MG17 nuclease.
  • SEQ ID NOs: 696-698 show the full-length peptide sequences of MG18 nucleases.
  • SEQ ID NOs: 700-702 show the nucleotide sequences of MG18 tracrRNAs.
  • SEQ ID NOs: 769-771 show the nucleotide sequences of MG18 CRISPR repeats.
  • SEQ ID NOs: 957-960 show the nucleotide sequences of sgRNAs engineered to function with an MG 18 nuclease.
  • MG46 [0175] SEQ ID NOs: 975-981 show the full-length peptide sequences of MG46 nucleases.
  • SEQ ID NOs: 703-709 show the nucleotide sequences of MG46 tracrRNAs.
  • SEQ ID NOs: 772-778 show the nucleotide sequences of MG46 CRISPR repeats.
  • SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972 show the nucleotide sequences of sgRNAs engineered to function with an MG46 nuclease.
  • SEQ ID NOs: 1265-1266 show the full-length peptide sequences of MG74 nucleases.
  • SEQ ID NOs: 1283-1392 and 1502-1509 show the nucleotide sequences of sgRNAs engineered to function with an MG21-1 nuclease in order to target the human HAO1 gene.
  • SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target the AAVS1 gene.
  • SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252 show the DNA sequences of AAVS1 target sites.
  • SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target TRAC.
  • SEQ ID Nos: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247 show the DNA sequences of TRAC target sites.
  • SEQ ID Nos: 579-592 and 1245 show the DNA sequences of human B2M target sites.
  • SEQ ID NOs: 593-625 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target the human HBB gene.
  • SEQ ID Nos: 626-658 show the DNA sequences of human HBB target sites.
  • SEQ ID NOs: 1083-1094 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target the human Albumin gene.
  • SEQ ID Nos: 1233-1244 show the DNA sequences of human Albumin target sites.
  • SEQ ID NOs: 1056-1081 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target human ATP7B.
  • SEQ ID NOs: 1206-1231 show the DNA sequences of human ATP7B target sites.
  • SEQ ID NOs: 1113-1122 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target the human PAH gene.
  • SEQ ID Nos: 1253-1262 show the DNA sequences of human PAH target sites.
  • SEQ ID NO: 61 shows the nucleotide sequence of an sgRNA engineered to function with an MG73-1 nuclease in order to target TRAC.
  • SEQ ID NOs: 63-69 and 263-302 show the nucleotide sequences of sgRNAs engineered to function with an MG89-2 nuclease in order to target TRAC.
  • SEQ ID NOs: 70-76 and 303-342 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 1011-1019, and 1050 show the nucleotide sequences of sgRNAs engineered to function with an MG87 nuclease in order to target TRAC.
  • SEQ ID Nos: 1161-1168 and 1200 show the DNA sequences of TRAC target sites.
  • SEQ ID NOs: 1020-1049 and 1051-1055 show the nucleotide sequences of sgRNAs engineered to function with an MG87 nuclease in order to target the AAVS1 gene.
  • SEQ ID NOs: 1169-1205 show the DNA sequences of AAVS1 target sites.
  • SEQ ID NOs: 1786-2045 show the nucleotide sequences of sgRNAs engineered to function with an MG21-1 nuclease in order to target the human ATXN2 gene.
  • SEQ ID NOs: 2051-2055 show the nucleotide sequences of sgRNAs engineered to function with an MG21-1 nuclease in order to target the mouse ATXN2 gene.
  • SEQ ID Nos: 1526-1785 show the DNA sequences of human ATXN2 target sites.
  • SEQ ID Nos: 2046-2050 show the DNA sequences of mouse ATXN2 target sites.
  • SEQ ID NOs: 1393-1493 show the nucleotide sequences of sgRNAs engineered to function with an MG23-1 nuclease in order to target the AAVS1 gene.
  • SEQ ID NOs: 1494-1501 and 1510-1525 show the nucleotide sequences of sgRNAs engineered to function with an MG23-1 nuclease in order to target TRAC.
  • SEQ ID NOs: 2056-2060 show the nucleic acid sequences of AAV cargo plasmids.
  • nucleotide refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides.
  • Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • the term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.
  • ATP adenosine triphosphate
  • UDP uridine triphosphate
  • CTP cytosine triphosphate
  • GTP guanosine triphosphate
  • deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives include, for example, [aS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moieties (e.g., fluorophores) or quantum dots.
  • Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.
  • Fluorescent labels of nucleotides include but are not limited fluorescein, 5- carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy- X-rhodamine (ROX), 4-(4 'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS).
  • FAM 5- carboxyfluorescein
  • JE 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein
  • rhodamine 6-carboxy
  • fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif;
  • nucleotide encompasses chemically modified nucleotides.
  • An exemplary chemically-modified nucleotide is biotin-dNTP.
  • biotinylated dNTPs include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14- dATP), biotin-dCTP (e.g., biotin- 11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin-11- dUTP, biotin- 16-dUTP, biotin-20-dUTP).
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • a polynucleotide can be exogenous or endogenous to a cell and/or exist in a cell-free environment.
  • the term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer.
  • Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino s, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • peptide refers to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e.g., domains).
  • amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component.
  • amino acid and amino acids refer to natural and non-natural amino acids, including, but not limited to, modified amino acids.
  • Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid.
  • amino acid includes both D-amino acids and L-amino acids.
  • operably linked refers to an arrangement of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element.
  • the effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element.
  • two genetic elements are operably linked if movement of the first element causes an activation of the second element.
  • a regulatory element which may comprise promoter and/or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.
  • a “functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence.
  • a biological activity of a DNA sequence includes its ability to influence expression in a manner attributed to the full-length sequence.
  • engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring.
  • An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein.
  • low sequence identity e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity
  • VPR and VP64 domains are synthetic transactivation domains.
  • Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring a new function or property.
  • An “engineered” system comprises at least one engineered component.
  • tracrRNA or “tracr sequence” means trans-activating CRISPR RNA.
  • tracrRNA interacts with the CRISPR (cr) RNA to form a guide nucleic acid (e.g., guide RNA or gRNA) that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid.
  • guide nucleic acid e.g., guide RNA or gRNA
  • a portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid.
  • the strand of a double- stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid is the complementary strand.
  • the strand of the double- stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand.
  • a guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.”
  • a guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.”
  • the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids.
  • a guide nucleic acid may comprise a segment referred to as a “nucleic acidtargeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.”
  • a nucleic acid-targeting segment can include a sub-segment referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment.”
  • the term “complex” refers to a joining of at least two components.
  • the two components may each retain the properties/activities they had prior to forming the complex or gain properties as a result of forming the complex.
  • the joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method.
  • Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof.
  • a complex comprises an endonuclease and a guide polynucleotide.
  • sequence identity or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm.
  • Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https://blast.ncbi.nlm.nih.gov) ; CLUSTALW with parameters of the Smith- Waterman homology search algorithm with parameters of a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAEET with parameters retree of 2 and maxiterations of 1000; Novafold with default parameters.
  • W wordlength
  • E expectation
  • variants of any of the enzymes described herein with one or more conservative amino acid substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide.
  • Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g. non-conserved residues) without altering the basic functions of the encoded proteins.
  • Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the endonuclease protein sequences described herein (e.g.
  • such conservatively substituted variants are functional variants.
  • Such functional variants can encompass sequences with substitutions such that the activity of critical active site residues of the endonuclease are not disrupted.
  • a functional variant of any of the proteins described herein lacks substitution of at least one of the residues predicted as essential. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of all of the residues predicted as essential.
  • Metagenomic sequencing from natural environmental niches that represent large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR/Cas systems known and speed the discovery of new oligonucleotide editing functionalities.
  • a recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX/CasY CRISPR systems from metagenomic analysis of natural microbial communities.
  • CRISPR/Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes.
  • CRISPR/Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally comprise two parts: (i) an array of short repetitive sequences (30-40 bp) separated by equally short spacer sequences, which encode the RNA-based targeting element; and (ii) ORLs encoding the Cas encoding the nuclease polypeptide directed by the RNA-based targeting element alongside accessory proteins/enzymes.
  • Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed (the PAM usually being a sequence not commonly represented within the host genome).
  • PAM protospacer-adjacent motif
  • Class 1 CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.
  • Type I CRISPR-Cas systems are considered of moderate complexity in terms of components.
  • the array of RNA-targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at repeat elements to liberate short, mature crRNAs that direct the nuclease complex to nucleic acid targets when they are followed by a suitable short consensus sequence called a protospacer-adjacent motif (PAM).
  • PAM protospacer-adjacent motif
  • This processing occurs via an endoribonuclease subunit (Cas6) of a large endonuclease complex called Cascade, which also comprises a nuclease (Cas3) that protein component of the crRNA- directed nuclease complex.
  • Cas 1 nucleases function primarily as DNA nucleases.
  • Type III CRISPR systems may be characterized by the presence of a central nuclease, known as Cas 10, alongside a repeat-associated mysterious protein (RAMP) that comprises Csm or Cmr protein subunits.
  • RAMP repeat-associated mysterious protein
  • the mature crRNA is processed from a pre- crRNA using a Cas6-like enzyme.
  • type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).
  • Type IV CRISPR-Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.
  • csfl highly reduced large subunit nuclease
  • csf3 two genes for RAMP proteins of the Cas5
  • csf2 Cas7
  • Class II CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.
  • Type II CRISPR-Cas systems are considered the simplest in terms of components.
  • the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature Cas9 enzyme loaded with both tracrRNA and crRNA.
  • Cas II nucleases are known as DNA nucleases.
  • the Cas9 effector has a characteristic structure, consisting of a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC- like nuclease domain.
  • the RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.
  • Type V CRISPR-Cas systems are characterized by a nuclease effector (Casl2) structure similar to that of Type ILCas9, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs; however, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems are capable of using the effector nuclease itself (Casl2) to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again known as DNA nucleases.
  • Casl2 nuclease effector
  • Type V enzymes e.g., Casl2a
  • Casl2a some Type V enzymes appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.
  • Type VI CRISPR-Cas systems are unique in that they appear to be the only class so far known as RNA-guided RNA endonucleases. Instead of RuvC-like domains, the single polypeptide effector of Type VI systems (Casl3) comprises two HEPN ribonuclease domains. Differing from both Type II and V systems, Type VI systems also appear to not need a tracrRNA for processing of pre-crRNA into crRNA. Similar to type V systems, however, some Type VI systems (e.g., C2C2) appear to possess robust single- stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of a target RNA.
  • C2C2C2C2C2 some Type VI systems (e.g., C2C2) appear to possess robust single- stranded nonspecific nuclease (ribonuclease) activity activated by the first crRNA directed cleavage of
  • pyogenes SF370 (ii) purified mature ⁇ 42 nt crRNA bearing a ⁇ 20 nt 5’ sequence complementary to the target DNA sequence desired to be cleaved followed by a 3’ tracr-binding sequence (the whole crRNA being in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA in vitro transcribed from a synthetic DNA template carrying a T7 promoter sequence, and (iv) Mg 2+ .
  • a later improved, engineered system involved the crRNA of (ii) joined to the 5’ end of (iii) by a linker (e.g., GAAA) to form a single fused synthetic guide RNA (sgRNA) capable of directing Cas9 to a target by itself.
  • a linker e.g., GAAA
  • sgRNA single fused synthetic guide RNA
  • Such engineered systems can be adapted for use in mammalian cells by providing DNA vectors encoding (i) an ORF encoding codon-optimized Cas9 (e.g., a Class 2, Type II Cas enzyme) under a suitable mammalian promoter with a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., TK pA signal); and (ii) an ORF encoding an sgRNA (having a 5’ sequence beginning with G followed by 20 nt of a complementary targeting nucleic acid sequence joined to a 3’ tracr-binding sequence, a linker, and the tracrRNA sequence) under a suitable Polymerase III promoter (e.g., the U6 promoter).
  • an ORF encoding codon-optimized Cas9 e.g., a Class 2, Type II Cas enzyme
  • a suitable mammalian promoter with a C-
  • the endonuclease is a Class 2, Type II endonuclease.
  • the endonuclease comprises a RuvC_III domain.
  • the endonuclease comprises an HNH domain.
  • the endonuclease is a double strand nuclease.
  • the endonuclease is catalytically dead.
  • the endonuclease is a double strand nuclease.
  • the endonuclease is modified. In some embodiments, the endonuclease is modified resulting in an endonuclease with nickase activity. In some embodiments, the modified endonuclease is a site-directed nickase
  • the endonuclease is a MG21 endonuclease. In some embodiments, the endonuclease is a MG23 endonuclease. In some embodiments, the endonuclease is a MG71 endonuclease. In some embodiments, the endonuclease is a MG73 endonuclease. In some embodiments, the endonuclease is a MG74 endonuclease. In some embodiments, the endonuclease is a MG89 endonuclease.
  • the endonuclease is a MG87 endonuclease. In some embodiments, the endonuclease is a MG88 endonuclease. In some embodiments, the endonuclease is a MG17 endonuclease. In some embodiments, the endonuclease is a MG 18 endonuclease. In some embodiments, the endonuclease is a MG46 endonuclease.
  • the engineered nuclease system is discovered through metagenomic sequencing.
  • the metagenomic sequencing is conducted on samples collected from various environments.
  • the environment is a human microbiome, an animal microbiome, an environment with high temperatures, an environment with low temperatures, or sediment.
  • the endonuclease is a MG21 endonuclease (i.e., SEQ ID NO: 47).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 47.
  • the endonuclease comprises a sequence having at least about 70% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 47.
  • the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 47.
  • the endonuclease is a MG23 endonuclease (i.e., SEQ ID NO: 48).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 48.
  • the endonuclease comprises a sequence having at least about 70% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 48.
  • the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 48.
  • the endonuclease is a MG71 endonuclease (i.e., SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 49-51, 679- 694, 983-1002, 1003-1010, and 1263.
  • the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 49-51, 679-694, 983- 1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003- 1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 49-51, 679- 694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003- 1010, and 1263.
  • the endonuclease is a MG73 endonuclease (i.e., SEQ ID NO: 51 and SEQ ID NO: 1264).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 51 or SEQ ID
  • the endonuclease comprises a sequence having at least about 70% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 51 or SEQ ID NO: 1264.
  • the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 51 or SEQ ID NO: 1264.
  • the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 51 or SEQ ID NO: 1264.
  • the endonuclease is a MG89 endonuclease (i.e., SEQ ID NO: 52).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 52.
  • the endonuclease comprises a sequence having at least about 70% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 52.
  • the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 52.
  • the endonuclease is a MG87 endonuclease (i.e., SEQ ID NOs: 659-660, 1158-1159, and 1267-1277).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277.
  • the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267- 1277. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277.
  • the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277.
  • the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277.
  • the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277.
  • the endonuclease is a MG88 endonuclease (i.e., SEQ ID NOs: 661-678 and 1278-1282).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 661-678 and 1278-1282.
  • the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 661-678 and 1278-1282.
  • the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 661-678 and 1278-1282.
  • the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 661-678 and 1278-1282.
  • the endonuclease is a MG17 endonuclease (i.e., SEQ ID NO: 659).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 659.
  • the endonuclease comprises a sequence having at least about 70% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to SEQ ID NO: 659.
  • the endonuclease comprises a sequence having at least about 95% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having 100% identity to SEQ ID NO: 659.
  • the endonuclease is a MG18 endonuclease (i.e., SEQ ID NOs: 696-698).
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 696-698.
  • the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 696-698.
  • the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 696-698.
  • the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 975-981.
  • the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 975-981.
  • the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 975-981.
  • the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 975-981.
  • the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 975-981. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 975-981. [0271] In some embodiments, the endonuclease is a MG74 endonuclease (i.e., SEQ ID NO: 1265 or SEQ ID NO: 1266).
  • the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266.
  • the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NO: 1265 or SEQ ID NO: 1266.
  • the endonuclease comprises a nuclear localization sequence (NLS).
  • NLS nuclear localization sequence
  • the NLS is at an N-terminus of the endonuclease.
  • the NLS is at a C-terminus of the endonuclease.
  • the NLS is at an N-terminus and a C-terminus of the endonuclease.
  • the NLS comprises a sequence of any one of SEQ ID NOs: 1-46, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-46.
  • the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 85% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 1- 46. In some cases, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 1-46.
  • the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 1-46. In some cases, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 1-46.
  • the engineered nuclease system disclosed herein comprises an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • an engineered guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • the guide RNAs comprise various structural elements including but not limited to: a spacer sequence which binds to the protospacer sequence (target sequence), a crRNA, and an optional tracrRNA.
  • the guide RNA comprises a crRNA comprising a spacer sequence.
  • the guide RNA comprises a tracrRNA or a modified tracrRNA.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 710-
  • the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 710-722. In some cases, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 710-722.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 726- 744.
  • the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 726-744. In some cases, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 726-744.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 745- 767.
  • the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 745-767. In some cases, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 745-767.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence having at least about 75% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 91% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence having at least about 93% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 96% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence having 100% identity to SEQ ID NO: 699.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 700-
  • the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 700-702. In some cases, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 700-702.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 70% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 75% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 80% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 85% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 90% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 91% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 92% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 93% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 94% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 95% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 96% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 97% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 98% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having at least about 99% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence comprising at least about 60-100 consecutive nucleotides having 100% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 703- 709.
  • the tracrRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 703-709.
  • the tracrRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 703-709. In some cases, the tracrRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system disclosed herein comprises an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • an engineered guide polynucleotide e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1137-1144.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1137-1144.
  • the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1137-1144.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1137-1144.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 1137-1144.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1153-1156.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1153-1156.
  • the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1153-1156.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1153-1156.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 1153-1156.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433- 2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969- 970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969- 970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 843-880 and 1145-1152.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 843-880 and 1145-1152.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 843-880 and 1145-1152.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 843-880 and 1145-1152.
  • the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 881-926.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 881-926.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to SEQ ID NO: 973 or SEQ ID NO: 974.
  • the engineered guide polynucleotide comprises a sequence having at least about 85% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to SEQ ID NO: 973 or SEQ ID NO: 974.
  • the engineered guide polynucleotide comprises a sequence having at least about 93% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to SEQ ID NO: 973 or SEQ ID NO: 974.
  • the engineered guide polynucleotide comprises a sequence having at least about 97% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to SEQ ID NO: 973 or SEQ ID NO: 974.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 957-960.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 957-960.
  • the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 957-960.
  • the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 957-960.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 957-960.
  • the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity to any one of SEQ ID NOs: 943-944, 951-956, 965-968, and 971-972.
  • the engineered guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the engineered guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter-nucleoside linkers of the engineered guide polynucleotide, or contiguous nucleotide sequence thereof, are modified.
  • the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
  • the engineered guide polynucleotide comprises modifications to a ribose sugar or nucleobase.
  • the engineered guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA.
  • the modification is within the ribose ring structure.
  • Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g., UNA).
  • the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids.
  • the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example peptide nucleic acids (PNA) or morpholino nucleic acids.
  • the engineered guide polynucleotide comprises one or more modified sugars.
  • the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides.
  • substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof.
  • nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides.
  • a 2’ sugar modified nucleoside in some embodiments, is a nucleoside that has a substituent other than -H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2’ substituted nucleosides and LNA (2’ -4’ biradical bridged) nucleosides.
  • 2’-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA (MOE), 2’- amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleosides.
  • the modification in the ribose group comprises a modification at the 2’ position of the ribose group.
  • the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O-methyl, 2’ -fluoro, 2’ -deoxy, and 2’-O-(2-methoxyethyl).
  • the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, the engineered guide polynucleotide comprises only modified sugars. In certain embodiments, the engineered guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2’-O- methoxyethyl group. In some embodiments, the engineered guide polynucleotide comprises both inter- nucleoside linker modifications and nucleoside modifications.
  • the engineered guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence.
  • the engineered guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.
  • the engineered guide polynucleotide is 30-250 nucleotides in length. In some embodiments, the engineered guide polynucleotide is more than 90 nucleotides in length. In some embodiments, the engineered guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the engineered guide polynucleotide is 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, or more than 240 nucleotides in length.
  • the engineered guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length.
  • the engineered guide polynucleotide comprises a tracrRNA.
  • the engineered guide polynucleotide comprises a guide nucleic acid (e.g., gRNA).
  • gRNA guide nucleic acid
  • a T means U (Uracil) in RNA and T (Thymine) in DNA.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 49-51, 679- 694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003- 1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003- 1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 710-722.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267- 1277 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 659-660, 1158- 1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 659-660, 1158- 1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 726-744.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 661-678 and 1278-1282 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 745-767.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 70% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 75% identity to SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 80% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 85% identity to SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 90% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 95% identity to SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 96% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 97% identity to SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 98% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising a sequence having at least about 99% identity to SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 659 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 699.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 700-702.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 75% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 80% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 703-709. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 85% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 90% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 703-709. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 95% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 96% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 703-709. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 97% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 98% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 703-709. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about at least about 99% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 703-709.
  • the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 975-981 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 703-709.
  • Described herein, in certain embodiments, is a cell comprising the systems described herein.
  • the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NS0), or human retinal cells), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or a Heliothis virescen
  • the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.
  • the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell, or derivative thereof.
  • nucleic acid sequences encoding an engineered nuclease system disclosed herein.
  • the nucleic acid encoding the engineered nuclease system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA.
  • the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.
  • the nucleic acid encoding the engineered nuclease system is delivered by a nucleic acid-based vector.
  • the nucleic acid-based vector is a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl-derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus.
  • cosmid e.g., pWE or sCos vectors
  • HAC human artificial chromosome
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosome
  • PAC Pl-derived artificial chromosomes
  • the nucleic acid-based vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT- 3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20- COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV- daGFP, pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF- CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal,pSF- OXB20-Fluc, pSF-OXB20,
  • the nucleic acid-based vector comprises a promoter.
  • the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof.
  • the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, pl9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof.
  • the promoter is a U6 promoter.
  • the promoter is a CAG promoter.
  • the promoter is encoded by a sequence of any one of SEQ ID NOs: 190-191, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity of any one of SEQ ID NOs: 190-191.
  • the nucleic acid-based vector is a virus.
  • the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus.
  • the virus is an alphavirus.
  • the virus is a parvovirus.
  • the virus is an adenovirus.
  • the virus is an AAV.
  • the virus is a baculovirus.
  • the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV- rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11,
  • the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV 11 or a derivative thereof.
  • the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof.
  • the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof.
  • the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof.
  • the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof.
  • the virus is AAV-DJ/8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.
  • the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV- 6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.
  • the nucleic acid encoding the engineered nuclease system is delivered by a non-nucleic acid-based delivery system (e.g., a non-viral delivery system).
  • a non-viral delivery system e.g., a liposome.
  • the nucleic acid is associated with a lipid.
  • the nucleic acid associated with a lipid in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • the nucleic acid is comprised in a lipid nanoparticle (LNP).
  • the fusion protein or genome editing system is introduced into the cell in any suitable way, either stably or transiently.
  • a fusion protein or genome editing system is transfected into the cell.
  • the cell is transduced or transfected with a nucleic acid construct that encodes a fusion protein or genome editing system.
  • a cell is transduced (e.g., with a virus encoding a fusion protein or genome editing system), or transfected (e.g., with a plasmid encoding a fusion protein or genome editing system) with a nucleic acid that encodes a fusion protein or genome editing system, or the translated fusion protein or genome editing system.
  • the transduction is a stable or transient transduction.
  • cells expressing a fusion protein or genome editing system or containing a fusion protein or genome editing system are transduced or transfected with one or more gRNA molecules, for example, when the fusion protein or genome editing system comprises a CRISPR nuclease.
  • a plasmid expressing a fusion protein or genome editing system is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lentivirus or AAV) or other methods known to those of skill in the art.
  • the gene editing system is introduced into the cell as one or more polypeptides.
  • delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and/or RNPs are known in the art, for example by electroporation or by cell squeezing.
  • Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • lipofection is described in e.g., U.S. Pat. Nos.
  • lipofection reagents are sold commercially (e.g., TransfectamTM, LipofectinTM and SF Cell Line 4D-Nucleofector X KitTM (Lonza)).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91/17424 and WO 91/16024.
  • the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).
  • the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.
  • Described herein, in certain embodiments, are methods for modifying a target nucleic acid comprising providing an engineered nuclease system disclosed herein.
  • the engineered nuclease system comprises an endonuclease and an engineered guide polynucleotide.
  • the target nucleic acid is double stranded.
  • the target nucleic acid is double stranded DNA.
  • the target nucleic acid is single stranded.
  • the methods are used to introduce a modification in the genome of a cell.
  • the modification is an insertion, deletion, or mutation.
  • the methods are used to introduce site-directed insertions, deletions, and/or mutations in the genome of a cell (for example an insertion and a mutation).
  • the methods are used in combination with a nucleic acid template to facilitate site- directed insertions into the genome of a cell.
  • the cell is a human cell.
  • the cell genome or a vector comprised in the cell is modified.
  • the cell genome is modified ex vivo.
  • the cell genome is modified in vivo.
  • the engineered guide polynucleotide targets a gene in a cell. In some embodiments, the engineered guide polynucleotide targets a gene in a mammalian cell. In some embodiments, the mammalian cell is a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, or a human cell.
  • the target gene is the HAO1 gene.
  • the gRNA comprises a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 47.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1283-1392 and 1502- 1509.
  • the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1283-1392 and 1502- 1509. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1283-1392 and 1502-1509.
  • the target gene is the AAVS1 gene.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 57-58, 101 - 119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 57-58, 101-119, 163- 181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 57-58, 101-119, 163- 181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 57-58, 101-119, 163- 181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.
  • the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098- 1102.
  • the gRNA hybridizes to the AAVS1 gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473- 484, 512-524, 552-564, and 1248-1252.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 59-60, 120-138, 182- 200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552- 564, and 1248-1252.
  • the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252.
  • the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252. In some embodiments, the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375- 406, 434-446, 473-484, 512-524, 552-564, and 1248-1252.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375- 406, 434-446, 473-484, 512-524, 552-564, and 1248-1252.
  • the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252.
  • the target gene is TRAC.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407- 413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 77-88, 139-150, 201- 212, 407-413, 447-453, 485-491, 525-531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 77-88, 139- 150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 77- 88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096- 1097. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525- 531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407- 413, 447-453, 485-491, 525-531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 77-88, 139-150, 201- 212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 77-88, 139- 150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 77- 88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.
  • the gRNA hybridizes to a TRAC sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414- 420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 70% identity to any one of SEQ ID NOs: 89-100, 151- 162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 75% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 80% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 85% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 90% identity to any one of SEQ ID NOs: 89-100, 151-162, 213- 224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 91% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 92% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 93% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 94% identity to any one of SEQ ID NOs: 89-100, 151-162, 213- 224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 95% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 96% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 97% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having at least about 98% identity to any one of SEQ ID NOs: 89-100, 151-162, 213- 224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes to a TRAC sequence having at least about 99% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the gRNA hybridizes to a TRAC sequence having 100% identity to any one of SEQ ID NOs: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.
  • the target gene is the B2M gene.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 565-578 and 1095.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 565-578 and 1095.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 565-578 and 1095.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 565-578 and 1095.
  • the gRNA hybridizes to a B2M gene sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 579-592 and 1245.
  • the gRNA hybridizes to a B2M sequence having at least about 70% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M sequence having at least about 75% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 80% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 85% identity to any one of SEQ ID NOs: 579-592 and 1245.
  • the gRNA hybridizes to a B2M gene sequence having at least about 90% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M sequence having at least about 91% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 92% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 93% identity to any one of SEQ ID NOs: 579-592 and 1245.
  • the gRNA hybridizes to a B2M gene sequence having at least about 94% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 95% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M gene sequence having at least about 96% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M sequence having at least about 97% identity to any one of SEQ ID NOs: 579-592 and 1245.
  • the gRNA hybridizes to a B2M sequence having at least about 98% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M sequence having at least about 99% identity to any one of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes to a B2M sequence having 100% identity to any one of SEQ ID NOs: 579-592 and 1245.
  • the target gene is the HBB gene.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 593-625.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 593-625.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 593-625.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 593-625. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 593-625.
  • the gRNA hybridizes to the HBB gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 626-658.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 626-658.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 626-658.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 626-658. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 626-658.
  • the target gene is the albumin gene.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1083-1094.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1083-1094.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1083-1094. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1083-1094.
  • the gRNA hybridizes to the albumin gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1233-1244.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1233-1244.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1233-1244.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1233-1244. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1233-1244.
  • the target gene is ATP7B.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1906-1931.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1906-1931.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1906-1931.
  • the gRNA hybridizes to ATP7B.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1206-1231.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1206-1231.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1206-1231.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1206-1231. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1206-1231.
  • the target gene is the PAH gene.
  • the gRNA is engineered to function with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263.
  • an MG71 endonuclease e.g., MG71-2; SEQ ID NO: 50
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1113-1122.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1113-1122.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1113-1122.
  • the gRNA hybridizes to the PAH gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1253-1262.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1253-1262.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1253-1262.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1253-1262.
  • the target gene is TRAC.
  • the gRNA is engineered to function with an MG73 endonuclease (e.g., MG73-1; SEQ ID NO: 51) comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 51 or SEQ ID NO: 1264.
  • an MG73 endonuclease e.g., MG73-1; SEQ ID NO: 51
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 61.
  • the gRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 61.
  • the gRNA comprises a sequence having at least about 75% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to SEQ ID NO: 61.
  • the gRNA hybridizes to TRAC.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 62.
  • the gRNA hybridizes to a sequence having at least about 70% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 91% identity to SEQ ID NO: 62.
  • the gRNA hybridizes to a sequence having at least about 92% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 96% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to SEQ ID NO: 62.
  • the gRNA hybridizes to a sequence having at least about 98% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to SEQ ID NO: 62. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to SEQ ID NO: 62. [0345] In some embodiments, the target gene is TRAC.
  • the gRNA is engineered to function with an MG89 endonuclease comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 52.
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 63-69 and 263-302.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 63-69 and 263-302.
  • the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 63-69 and 263-302.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 70-76 and 303-342.
  • the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 70-76 and 303-342.
  • the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1161-1169 and 1200.
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1020-1049 and 1051- 1055.
  • the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1020-1049 and 1051- 1055. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055.
  • the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055.
  • the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1020-1049 and 1051- 1055. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1020-1049 and 1051-1055.
  • the gRNA hybridizes to the AAVS1 gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1170-1205.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1170-1205.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1170-1205.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1170-1205.
  • the target gene is the ATXN2 gene.
  • the gRNA is engineered to function with an MG21 endonuclease comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 47.
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1786-2045.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1786-2045.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1786-2045.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1786-2045. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1786-2045.
  • the gRNA hybridizes to the ATXN2 gene.
  • the gRNA hybridizes to a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1526-1785.
  • the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 75% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1526-1785.
  • the gRNA hybridizes to a sequence having at least about 91% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 93% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 94% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1526-1785.
  • the gRNA hybridizes to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1526-1785. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 1526-1785.
  • the target gene is the AAVS1 gene.
  • the gRNA is engineered to function with an MG23 endonuclease comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 48.
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1393-1493.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1393-1493.
  • the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1393-1493.
  • the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1393-1493. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1393-1493. [0354] In some embodiments, the target gene is the AAVS1 gene.
  • the gRNA is engineered to function with an MG23 endonuclease comprising a sequence having at least at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 48.
  • the gRNA comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525.
  • the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1494-1501 and 1510- 1525.
  • the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525.
  • the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1494-1501 and 1510- 1525. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525.
  • the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1494-1501 and 1510-1525.
  • Example 1 Gene editing outcomes at the DNA level for TRAC and AAVS1 in K562 cells
  • 1.2 x 10 5 K562 cells were nucleofected with 500 ng mRNA/150 pmol guide, 250 ng mRNA/75 pmol guide, or 125 ng mRNA/37.5 pmol guide. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. Amplicons were sequenced and analyzed to measure gene editing (FIG. 3A).
  • 1.2 x 10 5 K562 cells were nucleofected with 500 ng mRNA/150 pmol guide. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. Amplicons were sequenced and analyzed to measure gene editing. Editing outcomes with MG71-2 engineered guides (scaffold 21) at exon 2 of the human B2M gene are shown in FIG. 5A. Editing outcomes with MG71-2 engineered guides (scaffold 21) at exon 3 of the human HBB gene are shown in FIG. 5B.
  • Putative Type II CRISPR effectors were identified by searching an extensive database. The resulting homologs were filtered to include those with an e-value ⁇ le' 5 , a length > 500 aa, and an associated CRISPR loci predicted using minCED. The effectors were dereplicated at 99% amino acid identity (AAI), globally aligned, and a phylogenetic tree was constructed.
  • AAI amino acid identity
  • ASR ancestral sequence reconstruction
  • Type II effectors were identified from the MG71, MG87, and MG88 families (SEQ ID NOs: 659-690). All the Type II effectors have the catalytic residues required for activity and range in length between 1,031 and 1,438 aa.
  • TracrRNAs were predicted. TracrRNAs (SEQ ID NOs: 699-767) and repeats (SEQ ID NOs: 768-836) were folded, trimmed, and connected with a tetraloop sequence GAAA, or TTCG if GAAA altered the fold. The effectors were screened with multiple sgRNA designs (SEQ ID NOs: 837-974) consisting of a short and long scaffold with multiple spacers varying in length (20, 22, and 24 nt) and GC content (40% and 67%).
  • Nucleases were expressed using 5-10 nM of a PCR-generated template. After expression, the nucleases were diluted 10-fold and incubated for 1 hour in a mixture containing 5 nM of an 8N protospacer adjacent motif (PAM) library plasmid and 50 nM sgRNA in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCh. The plasmid digest was cleaned using SPRI beads and eluted in TE buffer.
  • PAM protospacer adjacent motif
  • the digested PAM plasmids (15 nM) were blunt-end ligated to double-stranded adapter oligos (150 nM) with T4 ligase in IX T4 ligase buffer (NEB).
  • the ligated product was sequenced using 150 bp single read amplicon sequencing.
  • the resulting reads were filtered by a quality score >20.
  • PAMs were identified by mapping the reads to the PAM plasmid backbones requiring a perfect match. SeqLogos of the PAMs were generated using and PAM sequences were determined by the height of each nucleotide.
  • the cut site was identified by calculating the distance between the PAM and the ligated adapter.
  • the MG87 family of nucleases have the strongest preference in the 5th and 6th bases from the spacer (FIG. 10).
  • the MG88 family tend to have purine-rich PAMs in the second through fourth positions (FIG. 11).
  • Table 2 Summary of activity for MG71 family
  • Additional candidates were constructed using ancestral sequence reconstruction.
  • Ancestors MG71-42 and MG71-43 share 91.0% and 81.1% AAI with MG71-2, respectively.
  • Two out of three tested candidates showed robust activity with MG71-2, MG71-1, or MG 18-1 sgRNA (FIGs. 12A-12B, Table 7).
  • the resulting PAM of MG71-42 is similar to the natural metagenomic nuclease MG71-2.
  • the PAM of MG71-43 is more relaxed in vitro than that of MG71-2, but the MG71-2 PAM NNNACT is a subset of the observed PAM.
  • Example 8 Activity of MG71-2 gRNAs in mammalian cells
  • MG71-2 guides targeting a region of the human AAVS1 locus were designed with the MG71-2 in vitro PAM.
  • 50,000 K562 cells were nucleofected with 500 ng MG71-2 mRNA and 150 pmol chemically- synthesized guide RNA in a 96-well plate format and a cell-type specific program recommended by the manufacturer.
  • gDNA was extracted at 72 h post-nucleofection.
  • PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the target sequence. The amplicons were sequenced and analyzed to measure gene editing.
  • ASR ancestral sequence reconstruction
  • nucleases were expressed using 5-15 nM of a PCR- generated template with a T7 promoter. After expression, the nucleases were diluted 10-fold and digested for 1 hr in a mixture containing 5 nM protospacer adjacent motif (PAM) library plasmid and 50 nM sgRNA in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCh. The digest was cleaned using the SPRI beads and eluted in the TE buffer.
  • PAM protospacer adjacent motif
  • the digested PAM plasmids (15 nM) were blunt-end ligated to double-stranded adapter oligos (150 nM) with T4 ligase in IX T4 ligase buffer (NEB).
  • the ligated product was amplified with NGS adapters and sequenced using 300 bp single-read amplicon sequencing.
  • the resulting reads were filtered by a quality score >20.
  • the PAMs were identified by mapping the reads to the PAM plasmid backbones requiring a perfect match. SeqLogos of the PAMs were generated and the PAM sequences were determined by the height of each nucleotide.
  • the cut site was identified by calculating the distance between the PAM and the ligated adapter.
  • the target sequence of the guide RNA matched the sequence adjacent to the PAM library with a PAM design of TNNNNNA (5N PAM). Because the PAM region has a designed T in the first position, the exact nucleotide preference was not known, based on the WT MG71-2 protein’s PAM, it is assumed to be N. This is reported in the table below as a lower-case n since it is not empirically tested. These variants were all active in vitro (Table 8, FIG. 16):
  • ASRs ancestral sequence reconstructions
  • Example 10 In cell editing activity of MG nucleases at endogenous loci
  • the CDS codifying for nucleases MG87-21 (SEQ ID NO: 1158), MG87-70 (SEQ ID NO: 1159), MG71-2 (SEQ ID NO: 50), MG71-43 (SEQ ID NO: 694), MG21-1 (SEQ ID NO: 47), and MG23-1 (SEQ ID NO: 48) were cloned into a pUC19 plasmid including a RNA-pol T7 promoter, 5’ and 3’ UTRs, appropriate NLS sequences, and a 100 nt polyA tail (Table 10). To linearize the plasmid, 100 pg of plasmid was digested with Sapl.
  • the plasmid was purified with phenol/chloroform and precipitated with 70% ethanol.
  • the DNA pellet was resuspended in 20 pl of nuclease-free water.
  • 1 pg of linearized plasmid DNA was added to a 20 pl reaction containing IX reaction buffer (40 mM Tris-HCl pH 7.5, 16.5 mM MgCh, 50 mM NaCl, 2.5 mM Spermidine, 1 mM DTT) and 750 units of Hi-T7 RNA Polymerase. The reaction was incubated at 50°C for 1 hr. Transcribed mRNA was purified.
  • the in vitro transcription template could be generated via the PCR instead of plasmid digestion.
  • the ORF+UTRs was cloned into a plasmid without the promoter or a polyA tail and these were added via overhangs on the amplification primers. After clean-up via SPRI beads or gel extraction, this PCR product was used in the same in vitro transcription conditions.
  • the optimized RNA sequences are referenced in Table 10.
  • K562 cells were cultured in IMDM + 10% FBS at 37 °C and 5% CO2 per 100,000- 150,000 cells were nucleofected with the nuclease mRNA along with the indicated chemically- synthesized guide RNA (500 ng mRNA/150 pmol guide per well). 72 hr post-nucleofection, the cells were harvested and genomic DNA was extracted. The PCR primers appropriate for use in the NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA using DNA polymerase. Amplicons were sequenced and analyzed.
  • HBM Hepatocyte Basal Media
  • FBS Fetal Bovine Serum
  • 50,000 cells were nucleofected with nuclease mRNA along with the indicated chemically-synthesized guide RNA (500 ng mRNA/150 pmol guide per well).
  • 72 hr post-nucleofection the cells were harvested and the genomic DNA was extracted.
  • PCR primers appropriate for use in the NGS- based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA using DNA polymerase. Amplicons were sequenced and analyzed.
  • Hep3B cells were cultured in EMEM + 10% FBS + 1% penstrep at 37°C and 5% CO2. Once an adequate amount of cells had been cultured, 0.25% Trypsin-EDTA was added to the flasks to harvest the cells.
  • Harvested Hep3B cells were resuspended in buffer at a concentration of 50,000 cells/20uL. 50,000 cells/well were nucleofected with nuclease mRNA along with the indicated chemically-synthesized guide RNA (500 ng mRNA/150 pmol guide per well). 72 hr post-nucleofection, the cells were harvested and the genomic DNA extracted.
  • the PCR primers appropriate for use in the NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA using DNA polymerase. Amplicons were sequenced and analyzed.
  • MG87-70 (SEQ ID NO: 1107) was screened in K562 cells with 47 guides targeting sites with NNNNGT PAMs in AAVS1 or TRAC at two doses of guide, 150 pmol or 650 pmol.
  • MG71-2 and MG71-43 were screened for active guides to the intronic region of ATP7B, between exon 5 and exon 6 using an NNNACT PAM for guide design.
  • NNNACT PAM NNNACT PAM for guide design.
  • 24 guides were identified as active >1% in PHH cells and 26 guides were identified as active >1% in Hep3B cells (SEQ ID NOs: 1056-1082, FIGs. 20-21).
  • the two nucleases show similar levels of editing activity at these conditions.
  • MG71-2 and MG71-43 were screened for active guides to the intron 1 region of albumin, using an NNNACT PAM for guide design.
  • 10 guides were identified as active >1% in PHH cells for one or both nucleases (SEQ ID NOs: 1083-1092, FIG. 22).
  • 11 guides were designed only for MG71-43 using a less restrictive PAM of NNNRMY. Of these guides, 2 were active in PHH (SEQ ID NOs: 1093- 1094, FIG. 22).
  • the study utilized an off-target discovery method to identify potential off-targets of the nuclease.
  • Wild type-K562 cells were subcultured in a T75 flask using modified Improved Minimum Essential Medium (IMEM) with 10% fetal bovine serum (FBS) until confluent.
  • IMEM Improved Minimum Essential Medium
  • FBS fetal bovine serum
  • cells were harvested, washed in PBS, and 120K cells per 20uL were resuspended in the Nucleofector Solution (SF buffer). Cells were nucleofected with 500 ng of mRNA, 150pmol of guide, and 50 pmol of dsODN with K562 specific program and allowed to recover for 72 hrs.
  • dsODN was freshly prepared on the day of experiment by annealing (heat and slow cool) equimolar ratios of modified oligos in TE 8 buffer to make a 50
  • Fragmented adapter ligated DNA was size selected using magnetic beads and used as input material for the PCR reaction that enriches for fragments containing the dsODN sequence.
  • This PCR step was carried out in the form of two different reactions: one targeting the plus strand and the second targeting the minus strand, utilizing the primers that bind to the dsODN and end ligated adapters, thus enriching for fragments containing the dsODN.
  • the resulting DNA was quantified, normalized, and used for input in an indexing PCR which added Illumina sequencing adapters and as well as sample barcodes to each library.
  • the final libraries were pooled and purified before being quantified and sequenced. Reads were filtered to a max edit distance of 7 and read count of 10 to call off-targets.
  • MG71-2 and MG71-43 were screened with 8 guides that showed editing >80% (SEQ ID NOs: 1095-1102).
  • MG71-2 no detectable off targets were found for any of the guides.
  • MG71-43 five guides showed no detectable off-targets in 3 replicates.
  • two guides one off target was detected in one out of three replicates.
  • the remaining guide had two reproducible off-target sites in all three replicates, but still >98% of reads mapped to the on-target site (FIGs. 23A-23C).
  • MG87-70 (SEQ ID NO: 1107) was screened with 7 guides that showed editing >80% (SEQ ID NOs: 1023, 1025, 1029, 1030, 1042, 1045, 1049).
  • Four guides showed no detectable off-targets in 3 replicates, showing the high specificity of this nuclease for certain sites.
  • one off target was detected in a subset of the replicates, but still had >99% on-target reads.
  • the remaining guide gl_AAVSl_P2B3 had two reproducible off-target sites in all three replicates with overall on target reads ⁇ 80% of total reads (FIGs. 24A-24B).
  • Example 12 - Improved nucleases demonstrate high editing on therapeutically relevant loci in engineered mammalian cells
  • a subset of nucleases were tested in a pooled format to reduce the number of nucleofections required for screening.
  • 10 guides were pooled equimolar and used in transfection (15 pmol each, 150 pmol total).
  • the PAH pool was composed of SEQ ID NOs: 1113-1122. Protein MG71-88 showed activity at the engineered PAH site (FIGs. 25A).
  • Example 14 Human AAVS1 MG23-1 engineered guide RNA activity
  • Type II system 23-1 was tested with two spacer sequences (hAAVS 1 -231-G1 and hTRAC-23-l-H7) across eight spacer lengths (18-25 nt) on one chemically modified backbone pattern.
  • Type II system 21-1 was tested with one spacer sequence (hHAOl-21-l-Fl) across four spacer lengths (22-25 nt) and two different chemically modified backbones (78 and 157).
  • Type II system 71-2 was tested with one spacer (hTRAC-71-2-C2) across eight spacer lengths (18-25 nt) and two different chemically modified backbones (12 and 255).
  • Hep3B cells were nucleofected with 500 ng of nuclease mRNA and 65 pmol (231 nuclease), 30 pmol (368 nuclease), 2.5 pmol (211 nuclease), or 7.5 pmol (71-2 nuclease) of each engineered guide (SEQ ID NOs: 1486-1525) (50,000 cells per well in a 96-well plate).
  • Cells were harvested and genomic DNA extracted 72 hrs post- transfection.
  • PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the target sequence for each spacer. The amplicons were sequenced and analyzed to measure gene editing.
  • K562 cells were grown and passaged in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (v/v) fetal bovine serum at 37 °C with 5% CO2. Sequences for MG21-1 mRNA were codon optimized for human expression, then synthesized and cloned into a high copy kanamycin plasmid. Synthesized constructs encoding T7 promoter, UTRs, base editor ORF, and NLS sequences were digested with Hindll and BamHI, and ligated into a pUC19 plasmid backbone with T4 DNA ligase and lx reaction buffer.
  • IMDM Iscove's Modified Dulbecco's Medium
  • NLS sequences were digested with Hindll and BamHI, and ligated into a pUC19 plasmid backbone with T4 DNA ligase and lx reaction buffer.
  • the complete MG21-1 mRNA plasmid consists of an origin of replication, kanamycin resistance cassette, the synthesized construct, and an encoded polyA tail.
  • MG21-1 mRNA was synthesized via in vitro transcription (IVT) using the linearized mRNA plasmid. This plasmid was linearized by incubation at 37 °C for 4 hours with Spel enzyme.
  • a standard linearization reaction consists of a 50 uL reaction containing 10 ug pDNA, 40 units Spel, and lx reaction buffer. Linearized plasmid was purified and precipitated and resuspended in nuclease free water at an adjusted concentration of 500 ng/uL.
  • the IVT reaction to generate base editor mRNA was performed at 37 °C for 4 hr under the following conditions: 1.5 ug linearized plasmid; 4 mM ATP, CTP, GTP, Nl-methyl pseudo-UTP, and CleanCap AG 3’0ME; HiScribe Master Mix and lx transcription buffer. After 4 hr, IVT was stopped, and plasmid DNA was digested with the addition of 250 U/mL DNasel and incubated for 15 min at 37 °C. Purification of base editor mRNA was performed. Transcript concentration was determined by UV and further analyzed by capillary gel electrophoresis.
  • 1.0E+05 K562 cells were co-nucleofected with 500 ng of the above mRNAs and 150 pmol chemically- synthesized sgRNA targeting all exons of ATXN2.
  • Guide RNA spacer sequence SEQ ID NOs: 1526-1785 and full guide sequence SEQ ID NOs: 1786-2045 were evaluated.
  • Cells were grown for 3 days, harvested, and gDNA was extracted. Targeted regions for editing were amplified using DNA polymerase with primers appropriate for use with NGS- based DNA sequencing, and extracted DNA as the templates. PCR products were purified. Amplicons were sequenced and analyzed to measure gene editing.
  • NPC cells were cultured and passaged at 37 °C with 5% CO2. A total of 1.5 x 10 4 NPC cells were co-transfected with 200 ng of mRNA and chemically- synthesized sgRNA targeting ATXN2 at a molar ratio of 1:5 (mRNA:sgRNA).
  • gDNA genomic DNA
  • the target genomic regions were amplified from the extracted gDNA using DNA polymerase with primers designed for NGS-based sequencing. PCR products were purified. The amplicons were sequenced and analyzed to quantify gene editing efficiency.
  • human ATXN2 guides from Examples 16 and 17 with up to two mismatches to the mouse genome were converted to the corresponding mouse sequences.
  • These mouse surrogate guides were tested in Neuro-2a cells as synthetic guides.
  • the guides were transfected into the cells along with the corresponding editing system mRNA using nucleofection, and indels at the Atxn2 locus were measured.
  • Neuro-2a cells were cultured and passaged in the Dulbecco’s Modified Eagle’s Medium (supplemented with 10% FBS at 37 °C with 5% CO2. A total of 1.5 x 10 5 Neuro-2a cells were transfected using a serial dilution of MG21-1 mRNA and mouse surrogate Atxn2 guides via nucleofection. The highest concentration in the serial dilution was 500 ng of mRNA and 150 pmol of guide, followed by a 1:3 serial dilution series. Guide spacer sequence SEQ ID NOs: 2046-2050 and full guide sequence SEQ ID NOs: 2051-2055 were evaluated.
  • gDNA genomic DNA
  • the target genomic regions were amplified from the extracted gDNA using DNA polymerase with primers designed for the NGS-based sequencing.
  • the PCR products were purified.
  • the amplicons were sequenced and analyzed to quantify the gene editing efficiency.
  • Example 19 Validation of editing by mouse surrogate ATXN2 guides expressed from AAV cargo plasmids in mouse Neuro-2a cells
  • the AAV cargo plasmids were synthesized bearing a U6 promoter driving expression of the mouse surrogate guide RNAs created in Example 18, upstream of a CMV promoter driving V5 epitope-tagged MG21-1 bearing a synthetic poly- A (SpA). These plasmids were transfected into the mouse neuro-2a cells, and editing at the Atxn2 locus was measured as a function of plasmid amount.
  • AAV cargo constructs were designed to express MG21-1 and a guide RNA from a single AAV.
  • the payload was flanked by two, 141 bp inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • a U6 polymerase III promoter was placed upstream of the guide RNA sequence in the forward orientation at the 5’ end of the cargo. This was followed by a CMV promoter driving the MG21- 1 open reading frame.
  • MG21-1 was N-terminally tagged with a V5 epitope.
  • a synthetic polyadenylation sequence (SpA) followed MG21-1.
  • Neuro-2a cells were cultured and passaged in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS at 37 °C with 5% CO2. A total of 5 x 10 4 neuro-2a cells were transfected using a serial dilution of the AAV cargo plasmids via lipofection. SEQ ID NOs: 2056-2060 were evaluated. The highest plasmid concentration in the dilution series was 2 ug, followed by a 1:2 serial dilution.
  • gDNA genomic DNA
  • the target genomic regions were amplified from the extracted gDNA using DNA polymerase with the primers designed for the NGS-based sequencing.
  • the PCR products were purified.
  • the amplicons were sequenced and analyzed to quantify gene editing efficiency.
  • Example 20 Validation of editing by mouse surrogate ATXN2 guides expressed from AAV cargo plasmids in mouse Neuro-2a cells
  • Example 19 the same AAV cargo plasmids used in Example 19 were examined in an additional experimental repeat, employing the lower plasmid concentrations to maximize editing efficiency. These plasmids were transfected into the mouse neuro-2a cells, and editing at the Atxn2 locus was measured as a function of plasmid amount.
  • AAV cargo constructs were designed to express MG21-1 and a guide RNA from a single AAV.
  • the payload was flanked by two, 141 bp inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • a U6 polymerase III promoter was placed upstream of the guide RNA sequence in the forward orientation at the 5’ end of the cargo. This was followed by a CMV promoter driving the MG21- 1 open reading frame.
  • MG21-1 was N-terminally tagged with a V5 epitope.
  • a synthetic polyadenylation sequence (SpA) followed MG21-1.
  • Neuro-2a cells were cultured and passaged in the Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS at 37 °C with 5% CO2. A total of 5 x 10 4 Neuro-2a cells were transfected using a serial dilution of AAV cargo plasmids via lipofection. SEQ ID NOs: 2056- 2060 were evaluated. The highest plasmid concentration in the dilution series was 2 ug, followed by a 1:2 serial dilution. [0445] Cells were harvested after 3 days of culture, and genomic DNA (gDNA) was extracted. The target genomic regions were amplified from the extracted gDNA using DNA polymerase with primers designed for the NGS-based sequencing. The PCR products were purified. The amplicons were sequenced and analyzed to quantify the gene editing efficiency.
  • gDNA genomic DNA
  • the AAV cargo plasmids bearing V5-tagged MG21-1 and mouse Atxn2 guide RNAs under the control of promoters were transfected into mouse Neuro-2a cells.
  • the ability of these plasmids to knockdown ATXN2 protein levels was measured by semi- quantitative Western blot.
  • Neuro-2a cells were cultured and passaged in the Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS at 37 °C with 5% CO2. For transfection, a total of 2 x 10 5 Neuro-2a cells were transfected with each of five AAV plasmids (1 pg of DNA) containing different mouse surrogate A txn2 guides. SEQ ID NOs: 2056-2060 were evaluated. Three days after transfection, the cells were lysed in the RIPA buffer containing protease and phosphatase inhibitor cocktail. Of the cell lysate, 10 pg was loaded onto a 4-20% Tris-Glycine gel and transferred to the nitrocellulose membrane.
  • Blots were blocked with 5% milk, then probed with the antibodies against the V5 epitope (1:1000) and Ataxin2 (1:1000), detected using the fluorescent secondary antibody AF647-anti-mouse IgG (1:10000) and anti-rabbit IgG-IR800 (1:10,000) respectively.
  • the blots were reprobed with actin antibody (1:2000) as a reference control.
  • the highest knockdown level of mouse ATXN2 protein was 62% achieved by an AAV plasmid expressing guide P1F12 (FIG. 35). Protein knockdown of 44%, 41%, 38%, and 20% were achieved with AAV plasmids expressing guides P1A5, P2A1, P2G1, and P2F6, respectively.
  • Example 22 Packaging of full-lengh AAV cargo sequences into AAV9 as assessed by alkaline DNA gel electrophoresis
  • the packaging of full-length AAV cargos into AAV9 virions is demonstrated using alkaline DNA gel electrophoresis.
  • AAVs were produced using a transient triple co-transfection method involving a plasmid encoding adenoviral helper proteins, a plasmid encoding AAV rep protein and capsid proteins, and a plasmid encoding guide and MG21-1 flanked between two AAV2 ITRs. SEQ ID NOs: 2056-2060 were evaluated.
  • Viral Production Cells 2.0 were seeded at 1.5e6 cells/mL in BalanCD HEK293 medium supplemented with glutamine at 1:100 dilution.
  • the three plasmids were mixed at a 1:1:1 mass ratio with a total plasmid mass of 2 pg per mL of cell culture volume.
  • the plasmid mixture was diluted in 100 pL of IX PBS per mL of cell culture volume.
  • 1 pL of Rev IT AAV enhancer and 2 pL of TransIT Transfection Reagent per mL of cell culture volume were mixed into the diluted plasmid mixture, and incubated at room temperature for 30 minutes. The entire transfection mixture was added into the cells and incubated at 37 °C for 72 hrs prior to harvest.
  • the cells were harvested by centrifuging at 1500 g for 15 minutes at 4 °C and resuspended in a TMS buffer containing 50 mM of Tris-HCl, 2 mM MgCh, and 150 mM NaCl.
  • the cell pellets in the TMS buffer were subjected to physical lysis through 3 rapid freeze and thaw cycles, resulting in a crude viral lysate.
  • the crude viral lysate was treated with 100 U/mL of DENARASE® for 1 hr to digest exogenous nucleic acids.
  • the treated viral lysate was clarified by centrifugation at 12000 g for 60 minutes at 4 °C in two 30-minute steps, with the supernatant retained after each centrifugation step.
  • the clarified viral lysate was further purified via cesium chloride density gradient ultracentrifugation.
  • the alkaline gel was made by dissolving 1% agarose (w/v) in a solution of 30 mM NaCl and 2 mM EDTA in water, microwaved until a homogenous solution was formed, and then casted onto a gel casting tray.
  • the solidified gel was equilibrated in a running buffer containing 30 mM NaOH and 2 mM EDTA for 30 minutes.
  • the AAV samples were prepared by loading 2el0 vector genomes in 25 pL of 0.001% Pluronic F-68 in IX DPBS with calcium and magnesium at pH 7.10 and 5 pL of 6X alkaline loading dye. The solution was incubated at 95 °C for 5 minutes then cooled to 4 °C for 3 minutes.
  • the prepared samples were loaded onto the gel and ran at 50V for 210 minutes. Once the run had completed, the gel was placed in a neutralization solution consisting of 0.5 M Tris-HCl and 1 mM EDTA and incubated at room temperature for 1 hr on a rocking shaker. The neutralization solution was removed and replaced with a fresh neutralization solution and SYBR gold stain, and the gel was incubated for 1 hr at room temperature on a rocking shaker. The gel was imaged.
  • a neutralization solution consisting of 0.5 M Tris-HCl and 1 mM EDTA
  • Example 23- AAV9 virus particles bearing the MG21-1 editing system show effective transduction when injected into mouse olfactory bulb and cortex
  • Example 22 the AAV9 virus preparations shown in Example 22 were injected into the mouse olfactory bulb and motor cortex. The tissues were analyzed for the presence of viral genome copies to confirm transduction.
  • mice 6-8- week-old C57BL/6J mice were injected with AAV9 prepared in-house into both the olfactory bulb and Ml region of the motor cortex using a custom built stereotactic delivery system based on the RWD automated stereotaxic instrument (FIG. 37).
  • Ml injections consisted of a bilateral infusion of 750 nL of AAV9 infused at a rate of 5nl/second at coordinates +1.00 AP, +/-1.50 ML, -1.50 DV relative to Bregma.
  • Olfactory bulb injections consisted of a bilateral infusion of 250 nL of AAV9 infused at a rate of 5 nL/second and three target depths, coordinates +4.00 AP, +/- 0.70 ML, -2.5/-2.0/-1.5 DV relative to Bregma. A retention time of 5 minutes post injection was added to enhance absorption of the AAV into the tissue. Animals were sutured and allowed to recover until transferred back to their home cages. 2 weeks post injection, animals were euthanized via deep inhalation of isoflurane. Whole brains were dissected fresh, the olfactory bulb and the Ml regions were further dissected and processed for downstream nucleic acid extraction. Genomic DNA and mRNA was extracted from fresh tissue.
  • gDNA Purified genomic DNA
  • tissue titer of injected AAVs 5 ng of gDNA was added to a ddPCR reaction containing custom probes targeting the central region of the MG21-1 sequence multiplexed with a premade assay targeting the Cycl gene. Data were graphed. MG21-1 copies were normalized by Cycl reference copies to produce a normalized MG21-1 copies/cell value (FIG. 38). All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • Viral genome copies per cell varied across AAVs and injection sites, ranging from 2 to 63 copies per cell with an average of 8 copies per cell for olfactory bulb and 9 copies per cell for Ml motor cortex injections. The highest copy numbers were seen for P1F12 AAVs in the olfactory bulb, and P2G1 AAVs in the cortex.
  • Example 24- MG21-1 transcripts are detected in tissues injected with virus
  • Example 22 the AAV9 virus preparations shown in Example 22 were injected into the mouse olfactory bulb and motor cortex. The tissues were analyzed for the presence of MG21- 1 mRNA by ddPCR in order to confirm expression of the editing system.
  • mRNA was quantified. The mRNA was then used to produce cDNA by reverse transcription. To quantify the expression of AAVs in this study were loaded 5 ng of cDNA mRNA equivalent into a ddPCR reaction containing custom primers and probes targeting the central region of the MG21-1 sequence. This assay was additionally multiplexed targeting the Cycl gene. Data were graphed. MG21-1 copies were normalized by Cycl reference copies to produce a normalized MG21-1 copics/Cvc/ copies value (FIG. 39). All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • MG21-1 mRNA copies varied across AAVs and injection sites. The highest mRNA expression was seen for P1F12 AAV in the olfactory bulb, and P2G1 AAV in the cortex, consistent with the viral copy numbers obtained in FIG. 38.
  • the AAV9 virus preparations shown in Example 22 were injected into the mouse olfactory bulb and motor cortex.
  • the tissues were analyzed for the level of Atxn2 mRNA by ddPCR in order to gauge knockdown of target gene expression.
  • Atxn2 mRNA copies showed the greatest reduction in average mRNA expression in olfactory bulb samples injected with P1F12 AAV, and in cortex samples receiving the P2G1 AAV, consistent with the viral copy numbers and MG21-1 mRNA expression data obtained in FIG. 38 and FIG. 39, respectively.
  • Example 26- Indels within the ATXN2 gene are detectable in response to MG21-1 AAV delivery
  • gDNA Purified genomic DNA
  • NGS next-generation sequencing
  • the reaction used primers to specifically amplify a region surrounding the targeted cutsite.
  • Amplicons were then sent for NGS using 300xlbp reads.
  • Raw reads were filtered and aligned to a reference genome using a custom script.
  • Insertions and deletions were quantified using a custom script and graphed as a percentage of all filtered reads. Data were graphed. All individual data points are shown with the error bars denoting the average +/- standard error of the mean.
  • Indels within the mouse Atxn2 gene were highest in olfactory bulb samples injected with P1F12 AAV, and in cortex samples receiving the P2G1 AAV, consistent with the viral copy numbers, MG21-1 mRNA expression data, and Atxn2 transcript data obtained in FIGs. 38-41.
  • tissues harvested from the stereotactic injection of MG21-1 AAVs were analyzed for indels within the mouse Atxn2 gene.
  • Example 23 The methods are similar to as provided in Example 23.
  • Whole brains were dissected fresh and immediately frozen on dry ice, the olfactory bulb and the Ml regions were further dissected and processed for downstream protein extraction.
  • Protein was extracted by homogenizing frozen tissue in 2.5 mL tubes with ceramic beads in lysis buffer (IX HALT + lx PMSF in RIPA buffer) on a tissue homogenizer to create a homogenate.
  • the homogenate was then clarified by centrifugation at max speed at 4 °C for 10 minutes.
  • the clarified homogenate was then used in a Pierce BCA protein estimation assay.
  • Atxn2 protein levels appeared to be reduced in olfactory bulb samples injected with the P1F12 guide-expressing virus.
  • an AAV expressing MG21-1 with the P1F12 guide and an AAV expressing eGFP were co-injected into the mouse olfactory bulb.
  • the tissue was sectioned and Atxn2 protein visualized by immunofluorescence to detect knockdown.
  • V5 and eGFP expression were used to delineate the injected area.
  • mice 6-8- week-old C57BL/6J mice were injected with an AAV9 mixture consisting of MG21-1 P1F12 and Ula-EGFP prepared in house into the olfactory bulb using a custom built stereotactic delivery system based on the RWD automated stereotaxic instrument.
  • Olfactory bulb injections consisted of a bilateral infusion of 250 nL of AAV9 infused at a rate of 5nL/second and three target depths, coordinates +4.00 AP, +/- 0.70 ML, -2.5/-2.0/-1.5 DV relative to Bregma.
  • a retention time of 5 minutes post injection was added to enhance absorption of the AAV into the tissue. Animals were sutured and allowed to recover until transferred back to their home cages.
  • mice were euthanized via deep inhalation of isoflurane.
  • the animals were first perfused transcardially with 10 mL PBS followed by 10 mL 4% paraformaldehyde.
  • Whole brains were dissected and then cryoprotected in a solution of 30% sucrose prepared in PBS.
  • Cryoprotected whole brains were frozen in OCT, mounted and then sliced into 40 pm coronal sections using a Leica cryostat. Free-floating sections were blocked, permeabilized and then stained overnight at 4 °C with antibodies against Atxn2 (1:100) V5 (1:200) and GFP (1:1000). Stained sections were washed 3 times with 0.01% PBST and then incubated at room temperature for 2 hrs for secondary staining using Alexa- Fluor secondary antibodies. Finally, sections were washed 3 more times with 0.01% PBST and mounted on glass slides sealed with Fluoromount-G. Mounted slides were imaged. 40 pm Z-stacks were acquired by manually setting laser power and sensor gain to maximize signal-to-noise ratio for each channel. Data was then reconstructed for visualization and exported (FIG. 43). Scale bars represent 70 pm.
  • Atxn2 was seen to be expressed in a prominent ring within the olfactory bulb. A loss in Atxn2 expression appeared to correlate with the injected mixture of AAVs, as inferred by V5 and eGFP expression. This result suggested that the AAV expressing MG21-1 and guide P1F12 was capable of knocking down Atxn2 protein expression in vivo.
  • Example 29- Stereotactic injection of an AAV encoding MG21-1_P1F12 guide shows ATXN2 protein knockdown by imaging
  • an AAV expressing MG21-1 with the P1F12 guide and an AAV expressing eGFP were co-injected into the mouse olfactory bulb.
  • the tissue was sectioned and Atxn2 protein visualized by immunofluorescence to detect knockdown.
  • V5 and eGFP expression were used to delineate the injected area. This was a higher magnification of the images shown in Example 28.
  • mice 6-8 week old C57BL/6J mice were injected with an AAV9 mixture consisting of MG21-1 P1F12 and Ula-EGFP prepared in house into the olfactory bulb using a custom built stereotactic delivery system based on the RWD automated stereotaxic instrument.
  • Olfactory bulb injections consisted of a bilateral infusion of 250 nL of AAV9 infused at a rate of 5 nL/second and three target depths, coordinates +4.00 AP, +/- 0.70 ML, -2.5/-2.0/-1.5 DV relative to Bregma.
  • a retention time of 5 minutes post injection was added to enhance absorption of the AAV into the tissue. Animals were sutured and allowed to recover until transferred back to their home cages.
  • mice were euthanized via deep inhalation of isoflurane.
  • the animals were first perfused transcardially with 10 mL PBS followed by 10 mL 4% paraformaldehyde.
  • Whole brains were dissected and then cryoprotected in a solution of 30% sucrose prepared in PBS.
  • Cryoprotected whole brains were frozen in OCT, mounted and then sliced into 40 pm coronal sections using a Leica cryostat. Free-floating sections were blocked, permeabilized and then stained overnight at 4 °C with antibodies against Atxn2 (1:100) V5 (1:200) and GFP (1:1000). Stained sections were washed 3 times with 0.01% PBST and then incubated at room temperature for 2 hrs for secondary staining using Alexa- Fluor secondary antibodies. Finally, sections were washed 3 more times with 0.01% PBST and mounted on glass slides sealed with Fluoromount-G. Mounted slides were imaged on at 20X magnification. 40 pm Z-stacks were acquired by manually setting laser power and sensor gain to maximize signal-to-noise ratio for each channel. Data was then reconstructed for visualization and exported (FIG. 44). Scale bars represent 70 pm.
  • Example 28 provides a higher magnification view of the samples presented in Example 28. Atxn2 was seen to be expressed in a prominent ring within the olfactory bulb. A loss in Atxn2 expression appeared to correlate with the injected mixture of AAVs, as inferred by V5 and eGFP expression. This result suggests that the AAV expressing MG21-1 and guide P1F12 was capable of knocking down Atxn2 protein expression in vivo.
  • CRISPR systems The specificity of CRISPR systems depends not only on the sequences of guide RNA (gRNA) and target DNA, but also the Cas enzyme that is being used.
  • Lentivirus was used to deliver a library of dual-targets oligos, one containing a single mismatch in the spacer or PAM compared to the on-target, which are then edited in 96-well format by nucleofection with mRNA or RNP with chemically modified guides.
  • the mismatch tolerance obtained from the ratio of off:on target editing for each dual target oligo, was plotted by guide and highlights the guide- intrinsic mismatch tolerance observed.
  • a 96-well mismatch tolerance assay was developed (FIG. 45) in which cells were transduced with a library of dual-targets, one containing a single mismatch in the spacer or PAM compared to the on-target and edited by nucleofection to directly compare the editing efficiencies observed at both mismatched and on-target sequences using chemically modified guides.
  • RNAs that represented a range of GC contents and percent indels were chosen for use in this assay (FIGs. 46A and 46B.
  • An oligonucleotide library was generated from 49 guides, 21 guides for MG21-1 (SEQ ID NOs: 2062-2082) and 28 guides for MG71-2 (SEQ ID NOs: 2083-2110) to investigate their on- and off-target effects.
  • Each library member contained two targets: an on-target sequence and an off-target sequence with mismatches systematically introduced at every position within both the protospacer adjacent motif (PAM) and the spacer region.
  • PAM protospacer adjacent motif
  • control oligonucleotides were generated to simulate four possible editing events: cleavage at the on-target, cleavage at the off-target, cleavage at both targets, and no cleavage.
  • the library construct contains of the following components ordered left to right: a 20-bp left primer adapter sequence, an 18-bp barcode 1, a 24- 32 bp off-target sequence, a 28 bp linker segregating the two target sequences, a 24-32 bp on- target sequence, an 18-bp barcode 2, and an 20-bp right primer adapter sequence.
  • the lentilibrary transduced cells (IxlO 6 ) were edited via nucleofection of either mRNA and guide (2500 ng mRNA, 1000 pmol guide) or RNP (300 pmol of protein complexed with 500 pmol of sgRNA).
  • heatmaps were created to illustrate the protospacer base preference at each mismatch position for different nucleases.
  • the heatmaps were generated by grouping the data points by nuclease, mismatch location, and mismatch base, and then calculating the mean off-on ratio and 95% confidence interval for each group.
  • the resulting data was pivoted to create a matrix format suitable for heatmap visualization via seaborn, with color coding to indicate the mismatch tolerance at each base and position.
  • Editing controls were generated to characterize the editing outcomes of a dual-target library. The editing at left or right end was turned “OFF” by modifying the native PAM sequence (“ON”) associated with each on-target sequence. Editing observed across the four types of editing controls, no editing (OFF/OFF), left-end editing (OFF/ON, right-end editing, and both- end editing (ON/ON) were plotted in (FIG. 48) and show editing can be controlled through PAM inactivation and indels correctly assigned to the left or right end target.
  • FIG. 51 shows the single mismatch tolerance profile observed with 7 MG71-2 guides of 24-nucleotide length targeting the HBB locus, and when the two MG71-2 single mismatch tolerance profiles are overlaid on top of one another they show a high degree of similarity (FIG. 52).
  • Both Type II enzymes, MG71-2 and MG21-1 demonstrated lowest tolerance for single nucleotide mismatches at PAM proximal sites, increasing in tolerance moving distally from PAM.
  • PAM preference scores were calculated by converting mismatch tolerance scores and their respective off-target PAMs into information-based representation matrices using logomaker.
  • the PAM preferences for MG71-2 across both 22nt and 24nt spacers are represented as seqlogos in FIG. 54 depicting a consensus PAM of nnnACTnn.
  • the PAM preferences for MG21-1 across is represented as a seqlogo in FIG. 55 depicting a consensus PAM of RnGMAR.
  • FIG. 56 depicts the protospacer base preferences for MG71-2, and demonstrates slightly different protospacer base preferences for mismatches depending on the spacer length.
  • FIG. 57 depicts the protospacer base preferences for MG21-1.

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Abstract

La présente invention concerne des enzymes endonucléases ayant des caractéristiques de domaine distinctives, ainsi que des procédés d'utilisation de telles enzymes ou de variants de celles-ci.
PCT/US2024/056299 2023-11-16 2024-11-15 Enzymes à domaines ruvc Pending WO2025106934A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020168291A1 (fr) * 2019-02-14 2020-08-20 Metagenomi Ip Technologies, Llc Enzymes ayant des domaines ruvc
US20220235340A1 (en) * 2019-05-20 2022-07-28 The Broad Institute, Inc. Novel crispr-cas systems and uses thereof
WO2022232638A2 (fr) * 2021-04-30 2022-11-03 Metagenomi, Inc. Enzymes à domaines ruvc

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020168291A1 (fr) * 2019-02-14 2020-08-20 Metagenomi Ip Technologies, Llc Enzymes ayant des domaines ruvc
US20220235340A1 (en) * 2019-05-20 2022-07-28 The Broad Institute, Inc. Novel crispr-cas systems and uses thereof
WO2022232638A2 (fr) * 2021-04-30 2022-11-03 Metagenomi, Inc. Enzymes à domaines ruvc

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