Enzymes with RUVC domains

Abstract

The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.

Description

ENZYMES WITH RUVC DOMAINSCROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 599,932 filed November 16, 2023, which is incorporated by reference in its entirety herein.SUMMARY

[0002] Described herein, in certain embodiments, are 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.

[0003] In some embodiments, 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.

[0004] In some embodiments, 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.

[0005] In some embodiments, the engineered guide polynucleotide comprises a crRNA and a tracrRNA.

[0006] In some embodiments, 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.

[0007] In some embodiments, 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.

[0008] In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid.

[0009] In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid.

[0010] In some embodiments, the engineered guide polynucleotide is RNA.

[0011] In some embodiments, the endonuclease is not a Cas9 endonuclease.

[0012] In some embodiments, the endonuclease has less than 80% identity to a Cas9 endonuclease.

[0013] In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide.

[0014] In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.

[0015] Described herein, in certain embodiments, are 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.

[0016] Described herein, in certain embodiments, are 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.

[0017] Described herein, in certain embodiments, are 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, 961-962, 969-970, 1906-1931, 1083-1094, 1096-1102, 1113-1122, 1123-1137, and 2433-2434.

[0018] Described herein, in certain embodiments, are 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.

[0019] Described herein, in certain embodiments, are 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.

[0020] Described herein, in certain embodiments, are 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.

[0021] Described herein, in certain embodiments, are 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.

[0022] Described herein, in certain embodiments, are 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.

[0023] Described herein, in certain embodiments, are 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.

[0024] In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid.

[0025] In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid.

[0026] In some embodiments, the engineered guide polynucleotide is RNA.

[0027] In some embodiments, the endonuclease is not a Cas9 endonuclease.

[0028] In some embodiments, the endonuclease has less than 80% identity to a Cas9 endonuclease.

[0029] In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide.

[0030] In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.

[0031] In some embodiments, the endonuclease is fused to the engineered guide polynucleotide.

[0032] 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.

[0033] In some embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.

[0034] In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

[0035] In some embodiments, the modification is in vitro.

[0036] In some embodiments, the modification is in vivo.

[0037] In some embodiments, the modification is ex vivo.

[0038] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1161-1262, 1526-1785, and 2046-2050.

[0039] 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.

[0040] In some embodiments, the method further comprises selecting cells comprising the modification.

[0041] Provided here are 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.

[0042] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1283-1392 and 1502-1509.

[0043] Described herein, in certain embodiments, are methods of modifying ATPase copper transporting beta (ATP7B) comprising contacting ATP7B 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.

[0044] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1056-1081.

[0045] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1206-1231.

[0046] Described herein, in certain embodiments, are methods of modifying adeno-associated virus integration site 1 (AAVS1) 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.

[0047] In some embodiments, 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.

[0048] In some embodiments, 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.

[0049] 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.

[0050] In some embodiments, 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.

[0051] In some embodiments, 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.

[0052] 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 polynucleotideconfigured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

[0053] In some embodiments, the engineered guide polynucleotide comprises a sequence having any SEQ ID NO: 1083-1094.

[0054] In some embodiments, the target nucleic acid sequence comprises a sequence having SEQ ID NO: 1233-1244.

[0055] 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.

[0056] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 565-578 and 1095.

[0057] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 579-592 and 1245.

[0058] 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.

[0059] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 593-625.

[0060] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 626-658.

[0061] 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.

[0062] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1113-1122.

[0063] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1253-1262.

[0064] 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.

[0065] In some embodiments, the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1786-2045, and 2051-2055.

[0066] In some embodiments, the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1526-1785, and 2046-2050.

[0067] Described herein, in certain embodiments, are cells comprising the engineered nuclease systems disclosed herein.

[0068] In some embodiments, 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.

[0069] In some embodiments, 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.

[0070] In some embodiments, the cell is an engineered cell.

[0071] In some embodiments, the cell is a stable cell.

[0072] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0074] 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.

[0075] FIG. 2 depicts the gene editing outcomes at the DNA level for TRAC in K562 cells as described in Example 5.

[0076] 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.

[0077] FIG. 3B depicts analysis at the DNA level of second round of engineering for MG71-2 scaffold with guide hAAVSl C3.

[0078] 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.

[0079] 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.

[0080] 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).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] FIG. 13 depicts the gene editing outcomes at the DNA level (shown in % indel) for AAVS1 in K562 cells.

[0088] 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).

[0089] 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.

[0090] 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).

[0091] 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).

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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. The percent of dsODN reads coming from the desired target site was mapped for up to 3 replicates for nuclease MG71- 43. For 5 / 8 guides, all the reads passing analysis criteria come from the on-target site (FIG. 23B). The identified off-target sites for guide g6-MG71-2_AAVSl_H2 are shown relative to the on- target sequence (top, NNNRCY represents the PAM) (FIG. 23C). Dots denote matching position. The boxed bases represent changes from the on target. Two off targets are seen across the replicates. FIG. 23C discloses SEQ ID NOs: 2160-2163, 2160-2163 and 2160-2163, respectively, in order of appearance.

[0098] 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). The identified off-target sites for guide 87-70_sgRNA_PlB3 were shown relative to the on-target sequence (top, NNNNGT represents the PAM). Dots denote matching position (FIG. 24B). The boxed bases represent changes from the on target. Two off targets are seen across the replicates. FIG. 24B discloses SEQ ID NOs: 2164-2167, 2164-2167 and 2164-2167, respectively, in order of appearance.

[0099] 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. Guides were offset from each other by 1 nt. Each guide’s cut site lied within 10 nt of the disease mutation. Indel efficiency was evaluated by NGS.

[0100] FIG. 26 depicts human HAO1 MG21-1 engineered guide RNA activity.

[0101] FIG. 27 depicts human AAVS1 MG23-1 engineered guide RNA activity.

[0102] 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).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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). 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.

[0107] 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.

[0108] 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.

[0109] 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. 36 depicts 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.

[0111] 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.

[0112] 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. To determine the 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. MG21-1 copy numbers were normalized by CyCl reference copies to produce a normalized MG21-1 copies / cell value. All individual data points are shown with the error bars denoting the average + / - standard error of the mean.

[0113] FIG. 39 depicts MG21-1 transcripts are detected in the olfactory bulb and motor cortex after AAV9 injection. 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.

[0114] 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. 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] FIG. 47 depicts a schematic overview of the reference structure of dual-target oligo library member.

[0122] 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.

[0123] 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.

[0124] 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. 7 guides targeting 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0132] The Sequence Listing filed herewith provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions and systems according to the disclosure. Below are exemplary descriptions of sequences therein.

[0133] MG21

[0134] SEQ ID NO: 47 shows the full-length peptide sequence of an MG21 nuclease.

[0135] SEQ ID NOs: 1137-1144 show the nucleotide sequences of sgRNAs engineered to function with an MG21 nuclease.

[0136] MG23

[0137] SEQ ID NO: 48 shows the full-length peptide sequence of an MG23 nuclease.

[0138] SEQ ID NOs: 1153-1156 show the nucleotide sequences of sgRNAs engineered to function with an MG23 nuclease.

[0139] MG71

[0140] SEQ ID NOs: 49-51, 679-694, 983-1002, 1003-1010, and 1263 show the full-length peptide sequences of MG71 nucleases.

[0141] SEQ ID NOs: 53-54 show the peptide sequences of PAM-interacting domains of MG71 nucleases.

[0142] 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.

[0143] SEQ ID NOs: 710-722 show the nucleotide sequences of MG71 tracrRNAs.

[0144] SEQ ID NOs: 779-791 show the nucleotide sequences of MG71 CRISPR repeats.

[0145] 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. In SEQ ID NOs: 2433-2434, “n” can be any nucleotide.

[0146] MG73

[0147] SEQ ID NOs: 51 and 1264 show the full-length peptide sequence of a MG73 nuclease.

[0148] SEQ ID NO: 55 shows the peptide sequence of a PAM-interacting domain of a MG73 nuclease.

[0149] nnRnTTnn is a PAM sequence compatible with a MG73 nuclease.

[0150] MG89

[0151] SEQ ID NO: 52 shows the full-length peptide sequence of a MG89 nuclease.

[0152] SEQ ID NO: 56 shows the peptide sequence of a PAM-interacting domain of a MG89 nuclease.

[0153] nnnnCC is a PAM sequence compatible with a MG89 nuclease.

[0154] MG87

[0155] SEQ ID NOs: 659-660, 1158-1159, and 1267-1277 show the full-length peptide sequences of MG87 nucleases.

[0156] SEQ ID NOs: 726-744 show the nucleotide sequences of MG87 tracrRNAs.

[0157] SEQ ID NOs: 795-813 show the nucleotide sequences of MG87 CRISPR repeats.

[0158] SEQ ID NOs: 843-880 and 1145-1152 show the nucleotide sequences of sgRNAs engineered to function with an MG87 nuclease.

[0159] MG88

[0160] SEQ ID NOs: 661-678 and 1278-1282 show the full-length peptide sequences of MG88 nucleases.

[0161] SEQ ID NOs: 745-767 show the nucleotide sequences of MG88 tracrRNAs.

[0162] SEQ ID NOs: 814-836 show the nucleotide sequences of MG88 CRISPR repeats.

[0163] SEQ ID NOs: 881-926 show the nucleotide sequences of sgRNAs engineered to function with an MG88 nuclease.

[0164] MG17

[0165] SEQ ID NO: 695 shows the full-length peptide sequence of a MG17 nuclease.

[0166] SEQ ID NO: 699 shows the nucleotide sequence of a MG17 tracrRNA.

[0167] SEQ ID NO: 768 shows the nucleotide sequence of a MG17 CRISPR repeat.

[0168] SEQ ID NOs: 973-974 show the nucleotide sequences of sgRNAs engineered to function with an MG17 nuclease.

[0169] MG18

[0170] SEQ ID NOs: 696-698 show the full-length peptide sequences of MG18 nucleases.

[0171] SEQ ID NOs: 700-702 show the nucleotide sequences of MG18 tracrRNAs.

[0172] SEQ ID NOs: 769-771 show the nucleotide sequences of MG18 CRISPR repeats.

[0173] SEQ ID NOs: 957-960 show the nucleotide sequences of sgRNAs engineered to function with an MG 18 nuclease.

[0174] MG46

[0175] SEQ ID NOs: 975-981 show the full-length peptide sequences of MG46 nucleases.

[0176] SEQ ID NOs: 703-709 show the nucleotide sequences of MG46 tracrRNAs.

[0177] SEQ ID NOs: 772-778 show the nucleotide sequences of MG46 CRISPR repeats.

[0178] 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.

[0179] MG74

[0180] SEQ ID NOs: 1265-1266 show the full-length peptide sequences of MG74 nucleases.

[0181] HAO1 Targeting with MG21-1

[0182] 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.

[0183] MG71-2 AAVS1 Targeting

[0184] 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.

[0185] 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.

[0186] MG71-2 TRAC Targeting

[0187] 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.

[0188] 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.

[0189] MG71-2 B2M Targeting

[0190] SEQ ID NOs: 565-578 and 1095 show the nucleotide sequences of sgRNAs engineered to function with an MG71-2 nuclease in order to target the human B2M gene.

[0191] SEQ ID NOs: 579-592 and 1245 show the DNA sequences of human B2M target sites.

[0192] MG71-2 HBB Targeting

[0193] 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.

[0194] SEQ ID NOs: 626-658 show the DNA sequences of human HBB target sites.

[0195] MG71-2 Albumin Targeting

[0196] 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.

[0197] SEQ ID NOs: 1233-1244 show the DNA sequences of human Albumin target sites.

[0198] MG71-2 ATP7B Targeting

[0199] 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.

[0200] SEQ ID NOs: 1206-1231 show the DNA sequences of human ATP7B target sites.

[0201] MG71-2 PAH Targeting

[0202] 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.

[0203] SEQ ID NOs: 1253-1262 show the DNA sequences of human PAH target sites.

[0204] MG73-1 TRAC Targeting

[0205] SEQ ID NO: 61 shows the nucleotide sequence of an sgRNA engineered to function with an MG73-1 nuclease in order to target TRAC.

[0206] SEQ ID NO: 62 shows the DNA sequence of a TRAC target site.

[0207] MG89-2 TRAC Targeting

[0208] 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.

[0209] SEQ ID NOs: 70-76 and 303-342 show the DNA sequences of TRAC target sites.

[0210] MG87 TRAC Targeting

[0211] 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.

[0212] SEQ ID NOs: 1161-1168 and 1200 show the DNA sequences of TRAC target sites.

[0213] MG87 AAVS1 Targeting

[0214] 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.

[0215] SEQ ID NOs: 1169-1205 show the DNA sequences of AAVS1 target sites.

[0216] MG21-1 ATXN2 Targeting

[0217] 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.

[0218] 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.

[0219] SEQ ID NOs: 1526-1785 show the DNA sequences of human ATXN2 target sites.

[0220] SEQ ID NOs: 2046-2050 show the DNA sequences of mouse ATXN2 target sites.

[0221] MG23-1 AAVS1 Targeting

[0222] 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.

[0223] MG23-1 TRAC Targeting

[0224] 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.

[0225] AAV Cargo Plasmid

[0226] SEQ ID NOs: 2056-2060 show the nucleic acid sequences of AAV cargo plasmids.DETAILED DESCRIPTION

[0227] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0228] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0229] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0230] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA.

[0231] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0232] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0233] The term “nucleotide,” as used herein, 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. 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. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of 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). Specific examples of 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;FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein- 15 -dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6- dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15 -2'- dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY- TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein- 12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red- 12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically-modified nucleotide is biotin-dNTP. Non-limiting examples of 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).

[0234] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In a polynucleotide when referring to a T, 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.

[0235] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer 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). The terms also encompass an 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. The terms “amino acid”and “amino acids,” as used herein, 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. The term “amino acid” includes both D-amino acids and L-amino acids.

[0236] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer 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. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, 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.

[0237] 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.

[0238] The terms “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. For example, 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.

[0239] The term “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 orgRNA) that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid.

[0240] As used herein, a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid. 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.” If not otherwise specified, 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.”

[0241] As used herein, 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. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0242] The term “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, andthe 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.

[0243] Included in the current disclosure are variants of any of the enzymes described herein with one or more conservative amino acid substitutions. Such conservative 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. MG17, MG18, MG21, MG23, MG46, MG71, MG73, MG74, MG87, MG88, or MG89 family endonucleases described herein). In some embodiments, 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. In some embodiments, 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.

[0244] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (E), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)Overview

[0245] The discovery of new Cas enzymes with unique functionality and structure may offer the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. 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.

[0246] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as an adaptive immune system in microbes. In their natural context, 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). Depending on the exact function and organization of the system, CRISPR-Cas systems- l-are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity.

[0247] Class 1 CRISPR-Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV.

[0248] Type I CRISPR-Cas systems are considered of moderate complexity in terms of components. In Type I CRISPR-Cas systems, 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). 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.

[0249] 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. Like in Type I systems, the mature crRNA is processed from a pre- crRNA using a Cas6-like enzyme. Unlike type I and II systems, type III systems appear to target and cleave DNA-RNA duplexes (such as DNA strands being used as templates for an RNA polymerase).

[0250] 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.

[0251] Class II CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V and VI.

[0252] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, 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.

[0253] 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. Unlike Type II CRISPR-Cas systems, some Type V enzymes (e.g., Casl2a) 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.

[0254] 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.

[0255] Because of their simpler architecture, Class 2 CRISPR-Cas have been most widely adopted for engineering and development as designer nuclease / genome editing applications.

[0256] One of the early adaptations of such a system for in vitro use involved (i) recombinantly-expressed, purified full-length Cas9 (e.g., a Class 2, Type II Cas enzyme) isolated from .S'. 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) Mg2+. 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.

[0257] 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 acomplementary 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). MG Enzymes

[0258] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease and an engineered guide polynucleotide. In some embodiments, the endonuclease is a Class 2, Type II endonuclease. In some embodiments, the endonuclease comprises a RuvC_III domain. In some embodiments, the endonuclease comprises an HNH domain. In some embodiments, the endonuclease is a double strand nuclease. In some embodiments, the endonuclease is catalytically dead. In some embodiments, the endonuclease is a double strand nuclease. In some embodiments, 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

[0259] In some embodiments, 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. In some embodiments, 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.

[0260] In some embodiments, the engineered nuclease system is discovered through metagenomic sequencing. In some embodiments, the metagenomic sequencing is conducted on samples collected from various environments. In some embodiments, the environment is a human microbiome, an animal microbiome, an environment with high temperatures, an environment with low temperatures, or sediment.

[0261] In some embodiments, the endonuclease is a MG21 endonuclease (i.e., SEQ ID NO: 47). In some embodiments, 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. In some embodiments, 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 leastabout 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. In some embodiments, 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.

[0262] In some embodiments, the endonuclease is a MG23 endonuclease (i.e., SEQ ID NO: 48). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0263] In some embodiments, the endonuclease is a MG71 endonuclease (i.e., 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 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 leastabout 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0264] In some embodiments, the endonuclease is a MG73 endonuclease (i.e., SEQ ID NO: 51 and SEQ ID NO: 1264). In some embodiments, 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 IDNO: 1264. In some embodiments, 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 endonucleasecomprises a sequence having at least about 85% identity to SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, 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. In some embodiments, 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.

[0265] In some embodiments, the endonuclease is a MG89 endonuclease (i.e., SEQ ID NO: 52). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0266] In some embodiments, the endonuclease is a MG87 endonuclease (i.e., SEQ ID NOs: 659-660, 1158-1159, and 1267-1277). In some embodiments, the endonuclease comprises a sequence having at least about 30%, at least about 35%, at least about 40%, at least about 45%, atleast 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0267] In some embodiments, the endonuclease is a MG88 endonuclease (i.e., SEQ ID NOs: 661-678 and 1278-1282). In some embodiments, 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. In some embodiments, 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 leastabout 85% 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 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. In some embodiments, 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.

[0268] In some embodiments, the endonuclease is a MG17 endonuclease (i.e., SEQ ID NO: 659). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0269] In some embodiments, the endonuclease is a MG18 endonuclease (i.e., SEQ ID NOs: 696-698). In some embodiments, 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 about55%, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 696-698. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 696-698.

[0270] In some embodiments, the endonuclease is a MG46 endonuclease (i.e., SEQ ID NOs: 975-981). In some embodiments, 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. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 975-981. In some embodiments, 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. In some embodiments, 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 IDNOs: 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. In some embodiments, 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). In some embodiments, 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 NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 70% 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 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. In some embodiments, the endonuclease comprises a sequence having at least about 95% 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 96% 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 97% 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 98% 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 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.

[0272] In some embodiments, the endonuclease comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is at an N-terminus of the endonuclease. In some embodiments, the NLS is at a C-terminus of the endonuclease. In some embodiments, the NLS is at an N-terminus and a C-terminus of the endonuclease.

[0273] In some embodiments, 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. In some cases, 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. In some cases, 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.Table 1: Example NLS Sequences that can be used with Cas Effectors According to the DisclosureGuide Polynucleotides

[0274] In some embodiments, 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.

[0275] In some embodiments, 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. In some embodiments, the guide RNA comprises a crRNA comprising a spacer sequence. In some embodiments, the guide RNA comprises a tracrRNA or a modified tracrRNA.

[0276] In some cases, 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%, atleast 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. In some cases, 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. 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: 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. 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: 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. 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: 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. In some cases, 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.

[0277] In some cases, 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 leastabout 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. In some cases, 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. In some cases, 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. In some cases, 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.

[0278] In some cases, 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. In some cases, 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 sequencecomprising 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. 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: 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. 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: 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. In some cases, 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.

[0279] In some cases, 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. In some cases, 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 comprisesa 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. In some cases, 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. In some cases, 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.

[0280] In some cases, 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. In some cases, 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. 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: 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 toany 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 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. 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: 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. In some cases, 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.

[0281] In some cases, 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. In some cases, 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. In some cases, 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% identityto 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. In some cases, 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.

[0282] In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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 60-100consecutive 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. In some cases, 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.

[0283] In some cases, 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. In some cases, the tracrRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 699. In some cases, 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. In some cases, 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.

[0284] In some cases, 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%, atleast 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. In some cases, 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. 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: 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. 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: 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. In some cases, 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.

[0285] In some cases, 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 leastabout 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. In some cases, 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. In some cases, 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. In some cases, 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.

[0286] In some cases, 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. In some cases, 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 sequencecomprising 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. 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: 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. 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: 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. In some cases, 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.

[0287] In some cases, 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. In some cases, 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 comprisesa 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. In some cases, 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. In some cases, 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.

[0288] In some embodiments, 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.

[0289] 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 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. In some embodiments, 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. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1137-1144. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0290] 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 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. In some embodiments, 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. In some embodiments, 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 SEQID 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. In some embodiments, 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. In some embodiments, 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.

[0291] 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineeredguide 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% 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 99% 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 100% identity to any one of SEQ ID NOs: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.

[0292] 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 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. In some embodiments, 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. In some embodiments, the engineered guide polynucleotide comprises a sequence having at leastabout 90% 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 91% 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 92% 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 93% 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 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. In some embodiments, 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.

[0293] 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 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: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 881-926. In some embodiments, 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 SEQID 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. In some embodiments, 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. In some embodiments, 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 about50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or 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 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. In some embodiments, 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. In some embodiments, 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 polynucleotidecomprises 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. In some embodiments, 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.

[0294] 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 toany 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. In some embodiments, 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.

[0295] 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 about96% 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. In some embodiments, 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.

[0296] In some embodiments, 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. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0297] In some embodiments, the engineered guide polynucleotide comprises modifications to a ribose sugar or nucleobase. In some embodiments, 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. In some embodiments, 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). In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, 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.

[0298] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, 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. In some embodiments, substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substitutednucleosides. 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. Examples of 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. In some embodiments, the modification in the ribose group comprises a modification at the 2’ position of the ribose group. In some embodiments, 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).

[0299] In some embodiments, 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.

[0300] In some embodiments, 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. In some embodiments, 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.

[0301] In some embodiments, 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. In some embodiments, 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 about60, 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.MG Systems

[0302] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease comprising and an engineered guide polynucleotide. In some embodiments, the engineered guide polynucleotide comprises a tracrRNA. In some embodiments, the engineered guide polynucleotide comprises a guide nucleic acid (e.g., gRNA). In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0303] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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: 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. In some embodiments, 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. 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: 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. In someembodiments, 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. 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: 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. In some embodiments, 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. 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: 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. In some embodiments, 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.

[0304] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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: 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. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequencehaving 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. 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: 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. In some embodiments, 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. 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: 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. In some embodiments, 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. 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: 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. In some embodiments, 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.

[0305] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineerednuclease 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. In some embodiments, 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. 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: 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. In some embodiments, 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. 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: 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. In some embodiments, 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. 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: 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. In some embodiments, 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.

[0306] In some embodiments, 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. In some embodiments, 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 engineeredpolynucleotide comprising a sequence having at least about 80% 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0307] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 696-698 and an engineered polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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 IDNOs: 700-702. 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 700-702. 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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. 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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. 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: 696-698 and an engineered polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 700-702. In some embodiments, 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.

[0308] In some embodiments, 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. In some embodiments, 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. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at leastabout 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.Cells

[0309] Described herein, in certain embodiments, is a cell comprising the systems described herein.

[0310] In some embodiments, 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 retinalcells), 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 virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., a E. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, 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.

[0311] In some embodiments, 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.Delivery and Vectors

[0312] Disclosed herein, in some embodiments, are nucleic acid sequences encoding an engineered nuclease system disclosed herein.

[0313] In some embodiments, the nucleic acid encoding the engineered nuclease system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system is an RNA, for example a mRNA.

[0314] In some embodiments, the nucleic acid encoding the engineered nuclease system is delivered by a nucleic acid-based vector. In some embodiments, 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. In some embodiments, 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, pSF-Tac, pRI 101-AN DNA, pCambia2301,pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0315] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, 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. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, 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.

[0316] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, 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. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, 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.

[0317] In some embodiments, 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, AAV-HSC12, AAV- HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV- NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0318] In some embodiments, 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 someembodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Insome 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.

[0319] In some embodiments, 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.

[0320] In some embodiments, 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). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, 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. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0321] In some embodiments, the fusion protein or genome editing system is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein or genome editing system is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes a fusion protein or genome editing system. For example, 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. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In someembodiments, 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.

[0322] 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. 5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (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. In some embodiments, the delivery is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0323] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.Methods of Use

[0324] Described herein, in certain embodiments, are methods for modifying a target nucleic acid comprising providing an engineered nuclease system disclosed herein. In some embodiments, the engineered nuclease system comprises an endonuclease and an engineered guide polynucleotide. In some embodiments, the target nucleic acid is double stranded. In some embodiments, the target nucleic acid is double stranded DNA. In some embodiments, the target nucleic acid is single stranded.

[0325] In some embodiments, the methods are used to introduce a modification in the genome of a cell. In some embodiments, the modification is an insertion, deletion, or mutation. In some embodiments, 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). In some embodiments, the methods are used in combination with a nucleic acid template to facilitate site- directed insertions into the genome of a cell.

[0326] In some embodiments, the cell is a human cell. In some embodiments, the cell genome or a vector comprised in the cell is modified. In some embodiments, the cell genome is modified ex vivo. In some embodiments, the cell genome is modified in vivo.

[0327] In some embodiments, the engineered guide polynucleotide targets a gene in a cell. In some embodiments, the engineered guide polynucleotide targets a gene in a mammalian cell. Insome 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.

[0328] In some embodiments, the target gene is the HAO1 gene. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 70% 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0329] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 90% 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 91% 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0330] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) hybridizes to the AAVS1 gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 92% 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 93% 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 94% 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 95% 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 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% 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 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. In some embodiments, 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.

[0331] In some embodiments, the target gene is TRAC. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Insome embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0332] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0333] In some embodiments, the target gene is the B2M gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0334] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0335] In some embodiments, the target gene is the HBB gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 anyone 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. In some embodiments, 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. In some embodiments, 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.

[0336] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) hybridizes to the HBB gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 oneof 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. In some embodiments, 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.

[0337] In some embodiments, the target gene is the albumin gene. In some embodiments, 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. In some embodiments, 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: 1083-1094. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0338] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) hybridizes to the albumin gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0339] In some embodiments, the target gene is ATP7B. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1906-1931. In some embodiments, 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.

[0340] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) hybridizes to ATP7B. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0341] In some embodiments, the target gene is the PAH gene. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence havingat 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1113-1122. In some embodiments, 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.

[0342] In some embodiments, the gRNA (e.g., that functions with an MG71 nuclease) hybridizes to the PAH gene. In some embodiments, 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1253-1262. In some embodiments, thegRNA 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. In some embodiments, 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. In some embodiments, 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.

[0343] In some embodiments, the target gene is TRAC. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 70% identity to SEQ ID NO: 61. In some embodiments, 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 atleast 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. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having 100% identity to SEQ ID NO: 61.

[0344] In some embodiments, the gRNA (e.g., that functions with an MG73 nuclease) hybridizes to TRAC. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 atleast about 97% identity to SEQ ID NO: 62. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at least about 94% 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 95% 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 96% 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 97% 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 98% 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 99% identity to any one ofSEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 63-69 and 263-302.

[0346] In some embodiments, the gRNA (e.g., that functions with an MG89 nuclease) hybridizes to TRAC. In some embodiments, 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: 70-76 and 303-342. In some embodiments, 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 90% 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 91% 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 92% 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 93% 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 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 98% 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 99% identity to any one of SEQ ID NOs: 70-76 and 303-342. In some embodiments, the gRNA hybridizes to a sequence having 100% identity to any one of SEQ ID NOs: 70-76 and 303-342.

[0347] In some embodiments, the target gene is TRAC. In some embodiments, the gRNA is engineered to function with an MG87 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%, atleast 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. In some embodiments, 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: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 91% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 92% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 93% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 94% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having 100% identity to any one of SEQ ID NOs: 1011-1019 and 1050.

[0348] In some embodiments, the gRNA (e.g., that functions with an MG87 nuclease) hybridizes to TRAC. In some embodiments, 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 leastabout 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: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes to a sequence having at least about 70% 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 75% 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 80% 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 85% 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 90% 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 91% 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 92% 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 93% 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 94% 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 95% 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 96% 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 97% 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 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.

[0349] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, the gRNA is engineered to function with an MG87 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 any one of SEQ ID NOs: 659-660, 1158-1159, and 1267-1277. In some embodiments, 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%, atleast 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. In some embodiments, the gRNA comprises a sequence having at least about 70% 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 75% 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 80% 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 85% 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 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. In some embodiments, 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. In some embodiments, 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.

[0350] In some embodiments, the gRNA (e.g., that functions with an MG87 nuclease) hybridizes to the AAVS1 gene. In some embodiments, 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. In some embodiments, the gRNA hybridizes to a sequence having at least about 70% identity to any one of SEQ ID NOs: 1170-1205. In someembodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0351] In some embodiments, the target gene is the ATXN2 gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 gRNAcomprises 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. In some embodiments, 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. In some embodiments, 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.

[0352] In some embodiments, the gRNA (e.g., that functions with an MG87 nuclease) hybridizes to the ATXN2 gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, thegRNA 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. In some embodiments, 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.

[0353] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the gRNA comprises a sequence having at leastabout 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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% identityto any one of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, 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.EXAMPLESExample 1 - Gene editing outcomes at the DNA level for TRAC and AAVS1 in K562 cells

[0355] Nucleofection of MG71-2 mRNA along with the matching guide RNA (500 ng mRNA / 150 pmol guide) was performed into K562 cells (200,000). 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 and analyzed to measure gene editing (FIG. 1).Example 2 - Gene editing outcomes at the DNA level for TRAC in K562 cells

[0356] Nucleofection of MG89-2 mRNA along with the matching guide RNA (500 ng mRNA / 150 pmol guide) was performed into K562 cells (200,000). 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 and analyzed to measure gene editing (FIG. 2).Example 3 - Analysis of gene editing outcomes of the MG71-2 scaffold with guide hAAVSl C3 at the DNA level

[0357] 1.2 x 105K562 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). For analysis at the DNA level of second round of engineering for MG71-2 scaffold with guide hAAVSl C3, 1.2 x 105K562 cells were nucleofected with 500 ng mRNA / 150 pmol guide and processed for downstream steps as described above (FIG. 3B).Example 4 - Analysis of gene editing outcomes with original vs optimized MG71-2 scaffold at the DNA levels

[0358] 1.2 x 105K562 cells were nucleofected with 500 ng mRNA / 150 pmol guide and processed for downstream steps. 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 (FIG. 4).Example 5 - Analysis of gene editing outcomes with original vs optimized MG71-2 scaffold at the DNA levels

[0359] 1.2 x 105K562 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.Example 6 - In silico identification of Type II CRISPR effectors

[0360] In silico identification of Type II CRISPR effectors

[0361] 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.

[0362] Ancestral reconstruction ofMG71 nucleases

[0363] In order to generate further diversity of the MG71 family of Type II nucleases, ancestral sequence reconstruction (ASR) algorithms were used. ASR is a computational technique that uses existing protein sequences and the relationships inferred between them to reconstruct potential ancestral sequences from ancient organisms. This technique was used to reconstruct sequences from the MG71 family. For this analysis, 432 Type II nuclease sequences were aligned and a phylogenetic tree was built. The trees were rooted using two Type II-B sequences as outgroups. Sequence reconstruction was done. Insertions and deletions were identified manually for each reconstructed node. Four ancestral sequences were reconstructed with high confidence: MG71-40, MG71-41, MG71-42, MG71-43 (FIG. 6, SEQ ID NOs: 691-694).

[0364] Results

[0365] 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.Example 7 - sgRNA design and in vitro activity of MG71, MG73, MG74, MG87, and MG88 nucleases

[0366] sgRNA design

[0367] 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%).

[0368] In vitro activity assay

[0369] 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. 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.

[0370] Results

[0371] Multiple candidates from each family were active, demonstrating site-directed nuclease activity with their respective sgRNA designs. The cut sites and PAMs identified from the NGS data are summarized in Tables 2-6 and example PAM SeqLogos are in FIGs. 7-11. Each family has a variety of PAMs, demonstrating the versatility of these systems as potential gene editing agents. The nucleases derived from metagenomic data from the MG71 family, which includes the MG 18 and MG46 families, display a range of PAM specificities, with preference for an A in the 4th position (FIG. 7). The MG74, including MG17, family has A / C rich PAMs (FIG. 9). 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 familyTable 3: Summary of activity for MG73 familyTable 4: Summary of activity for MG74 familyTable 5: Summary of activity for MG87 familyTable 6: Summary of activity for MG88 family

[0372] 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.Table 7: Summary of activity for MG71 ASRs iNuclease |PAM |Cut site |Example 8 - Activity of MG71-2 gRNAs in mammalian cells

[0373] To further test the activity of 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.

[0374] Results

[0375] Several guides showed > 50% activity, further supporting the robust activities of the nuclease (FIG. 13).Example 9 - In vitro activity of Type II CRISPR systems with rational point mutations

[0376] Ancestral Sequence Reconstruction (ASR) to generate MG71 variants

[0377] To generate additional diversity of the MG71 family of Type II nucleases, ancestral sequence reconstruction (ASR) algorithms were used, as described in Example 6 above. For this analysis, 432 Type II nuclease sequences were aligned using MAFFT with parameters, a phylogenetic tree was built, and ancestral nodes were reconstructed. Eight ancestral nodes were reconstructed: MG71-88, MG71-89, MG71-90, MG71-91, MG71-92, MG71-95, MG71-96, MG71-97 (FIG. 14, SEQ ID NOs. 1003-1010).

[0378] In vitro activity assay

[0379] As described in Example 7 above, 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. 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 theheight of each nucleotide. The cut site was identified by calculating the distance between the PAM and the ligated adapter.

[0380] Results

[0381] Based on the Alphafold2 predicted structure of MG71-2 overlaid with the structure of SpCas9 (PDB ID: 4UN3), 16 residues within the first interaction shell of the PAM base pair (FIG. 15) were selected. To change the PAM sequence of MG71-2, the 16 residues were mutated so that positively charged residues were either installed or removed. Analogous residues were identified in MG71-43 (SEQ ID NO: 694) to generate four MG71-43 mutants. These were screened in vitro with the MG71-2 optimized sgRNA 71-2_C2D2_U40_22 (SEQ ID NO: 982). 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):Table 8: PAMs of single-point mutant variants of MG71 proteins

[0382] From these results, we see multiple residues can affect the PAM, with the strongest effect to the PAM specificity resulting from the mutations in residues K1352, V1353, and Q1210 in MG71-2 (protein SEQ ID NOs: 990, 993, and 994; PAM’s nNWRAT, nNNRMT, and nNNRYY) and the homologous residues Q1206 and K1348 in MG71-43 (protein SEQ ID NOs: 1000 and 1002; PAM’s nRNRCN and nRNRNT). In contrast other mutations in MG71-2, especially R1206A, L1351R, N1355R and N1356R variants (SEQ ID NOs: 985, 992, 996, and 997) does not lead to changes to the native PAM motif of the wild-type enzyme.

[0383] Additionally, eight ancestral sequence reconstructions (ASRs) were screened for activity in vitro with the same MG71-2 sgRNA. The PAMs for these variants are shown in Table 9 and FIG. 17. As has been observed with other nucleases of the MG71 family, we see a tendency for A or R in the fourth position and T or Y in the 6th position of the PAM.Table 9: PAMs of ASR variants of MG71 proteinsExample 10 - In cell editing activity of MG nucleases at endogenous loci

[0384] mRNA synthesis

[0385] 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. For in vitro transcription, 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. Alternatively, the in vitro transcription template could be generated via the PCR instead of plasmid digestion. In this case,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.Table 10: mRNA sequence information for different nucleases

[0386] Mammalian cell activity assays

[0387] 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.

[0388] Primary Human Hepatocytes were thawed and resuspended in Hepatocyte Basal Media (HBM) supplemented with HCM SingleQuots and 5% Fetal Bovine Serum (FBS). 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.

[0389] 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 concentrationof 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.

[0390] Results

[0391] 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.39 guides showed activity >2% in at least one of the screen conditions (SEQ ID NOs: 1011-1049, FIG. 18). The highest observed editing with the 150 pmol dose was 93.8%. Indel rates only increase slightly at the higher dose...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered nuclease system comprising: a) 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 b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.

2. The engineered nuclease system of claim 1, wherein 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.

3. The engineered nuclease system of claim 1, wherein 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.

4. The engineered nuclease system of any one of claims 1-3, wherein the engineered guide polynucleotide comprises a crRNA and a tracrRNA.

5. The engineered nuclease system of claim 4, wherein 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.

6. The engineered nuclease system of claim 4, wherein 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.

7. The engineered nuclease system of any one of claims 1-6, wherein the engineered guide polynucleotide is a single guide nucleic acid.

8. The engineered nuclease system of any one of claims 1-6, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

9. The engineered nuclease system of any one of claims 1-8, wherein the engineered guide polynucleotide is RNA.

10. The engineered nuclease system of any one of claims 1-9, wherein the endonuclease is not a Cas9 endonuclease.

11. The engineered nuclease system of any one of claims 1-10, wherein the endonuclease has less than 80% identity to a Cas9 endonuclease.

12. The engineered nuclease system of any one of claims 1-11, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.

13. The engineered nuclease system of any one of claims 1-11, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

14. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47; and b) 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.

15. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 48; and b) 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.

16. An engineered nuclease system comprising: a) 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 b) 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, 961-962, 969-970, 1906-1931, 1083-1094, 1096-1102, 1113-1122, 1123-1137, and 2433-2434.

17. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 51 or SEQ ID NO: 1264; and b) 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.

18. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NOs: 659, 660, 1158, 1159, and 1267-1277; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guidepolynucleotide 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.

19. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 661-678 and 1278-1282; and b) 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.

20. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 659; and b) 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.

21. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 696-698; and b) 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.

22. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 975-981; and b) 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.

23. The engineered nuclease system of any one of claims 14-22, wherein the engineered guide polynucleotide is a single guide nucleic acid.

24. The engineered nuclease system of any one of claims 14-22, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

25. The engineered nuclease system of any one of claims 14-24, wherein the engineered guide polynucleotide is RNA.

26. The engineered nuclease system of any one of claims 1-25, wherein the endonuclease is not a Cas9 endonuclease.

27. The engineered nuclease system of any one of claims 14-26, wherein the endonuclease has less than 80% identity to a Cas9 endonuclease.

28. The engineered nuclease system of any one of claims 14-27, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.

29. The engineered nuclease system of any one of claims 14-27, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

30. The engineered nuclease system of any one of claims 14-27, wherein the endonuclease is fused to the engineered guide polynucleotide.

31. A method for modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of any one of claims 1-30.

32. The method of claim 31, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving, the target nucleic acid sequence.

33. The method of any one of claims 31-32, wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

34. The method of any one of claims 31-33, wherein the modification is in vitro.

35. The method of any one of claims 31-33, wherein the modification is in vivo.

36. The method of any one of claims 31-33, wherein the modification is ex vivo.

37. The method of any one of claims 31-36, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1161-1262, 1526-1785, and 2046-2050.

38. A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of any one of claims 1-30.

39. The method of claim 38, further comprising selecting cells comprising the modification.

40. A method of modifying a hydroxyacid oxidase 1 (HAO1) gene comprising contacting the HAO1 gene using an engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47; and b) 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.

41. The method of claim 40, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1283-1392 and 1502-1509.

42. A method of modifying ATPase copper transporting beta (ATP7B) comprising contacting ATP7B using an engineered nuclease system comprising: a) 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 b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

43. The method of claim 42, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1056-1081.

44. The method of claim 42, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1206-1231.

45. A method of modifying adeno-associated virus integration site 1 (AAVS1) comprising contacting AAVS1 using an engineered nuclease system comprising: a) 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 b) 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.

46. The method of claim 45, wherein 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.

47. The method of claim 45, wherein 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.

48. A method of modifying T cell receptor alpha constant (TRAC) comprising contacting TRAC using an engineered nuclease system comprising: a) 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 b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guidepolynucleotide comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 710-722.

49. The method of claim 48, wherein 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.

50. The method of claim 48, wherein 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.

51. A method of modifying an albumin gene comprising contacting the albumin gene using an engineered nuclease system comprising: a) 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 b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

52. The method of claim 51, wherein the engineered guide polynucleotide comprises a sequence having any SEQ ID NO: 1083-1094.

53. The method of claim 48, wherein the target nucleic acid sequence comprises a sequence having SEQ ID NO: 1233-1244.

54. A method of modifying a beta-2-microglobulin (B2M) gene comprising contacting the B2M gene using an engineered nuclease system comprising: a) 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 b) 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.

55. The method of claim 54, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 565-578 and 1095.

56. The method of claim 54, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 579-592 and 1245.

57. A method of modifying a hemoglobulin subunit beta (HBB) gene comprising contacting the HBB gene using an engineered nuclease system comprising: a) 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; andb) 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.

58. The method of claim 57, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 593-625.

59. The method of claim 57, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 626-658.

60. A method of modifying a phenylalanine hydroxylase (PAH) gene comprising contacting the PAH gene using an engineered nuclease system comprising: a) 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 b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

61. The method of claim 60, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1113-1122.

62. The method of claim 60, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1253-1262.

63. A method of modifying an ataxin 2 (ATXN2) gene comprising contacting the ATXN2 gene using an engineered nuclease system comprising: c) an endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 47; and d) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

64. The method of claim 63, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NOs: 1786-2045, and 2051-2055.

65. The method of claim 63, wherein the target nucleic acid sequence comprises a sequence of any one of SEQ ID NOs: 1526-1785, and 2046-2050.

66. A cell comprising the engineered nuclease system of any one of claims 1-30.

67. The cell of claim 66, wherein the cell is a eukaryotic cell.

68. The cell of claim 66, wherein the cell is a mammalian cell.

69. The cell of claim 66, wherein the cell is an immortalized cell.

70. The cell of claim 66, wherein the cell is an insect cell.

71. The cell of claim 66, wherein the cell is a yeast cell.

72. The cell of claim 66, wherein the cell is a plant cell.

73. The cell of claim 66, wherein the cell is a fungal cell.

74. The cell of claim 66, wherein the cell is a prokaryotic cell.

75. The cell of claim 66, wherein 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.

76. The cell of claim 66, wherein the cell is an engineered cell.

77. The cell of claim 66, wherein the cell is a stable cell.

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