Enzymes with ruvc domain

Abstract

This disclosure provides endonucleases with distinctive structural domain features, and methods for using such enzymes or variants thereof.

Description

[0001] Cross-referencing

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 599,932, filed November 16, 2023, which is incorporated herein by reference in its entirety. Summary of the Invention

[0003] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 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 nuclease and hybridize with a target nucleic acid sequence.

[0004] In some embodiments, the endonuclease comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO: 47-52, 659-698, 983-1010, 1263-1282, 1158-1159 and 975-981.

[0005] In some embodiments, the endonuclease comprises a sequence having 90% sequence identity with any one of SEQ ID NO: 47-52, 659-698, 983-1010, 1263-1282, 1158-1159 and 975-981.

[0006] In some embodiments, the engineered guide polynucleotide comprises crRNA and tracrRNA.

[0007] In some embodiments, the tracrRNA comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 710-722, 726-744, 745-767, 699, 700-702, and 703-709.

[0008] In some embodiments, the tracrRNA comprises a sequence having 100% sequence identity with any one of SEQ ID NO: 710-722, 726-744, 745-767, 699, 700-702, and 703-709.

[0009] In some embodiments, engineered guide polynucleotides are single-guide nucleic acids.

[0010] In some embodiments, the engineered guide polynucleotide is a bidirectional guide nucleic acid.

[0011] In some embodiments, the engineered guide polynucleotide is RNA.

[0012] In some embodiments, the endonuclease is not the Cas9 endonuclease.

[0013] In some embodiments, the endonuclease has less than 80% identity with the Cas9 endonuclease.

[0014] In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide.

[0015] In some embodiments, the endonuclease is covalently linked to an engineered guide polynucleotide.

[0016] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 47; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1137-1144, 1283-1392, 1502-1509, and 1786-2045.

[0017] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 48; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 1153-1156, 1393-1493, 1494-1501, and 1510-1525.

[0018] In some embodiments, this document describes an engineered nuclease system comprising a nuclease having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide .... 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, 565-57 8. A sequence that has at least 80% sequence identity among any one of 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.

[0019] In some embodiments, an engineered nuclease system is described herein comprising: a nuclease comprising a sequence having at least 80% sequence identity with either SEQ ID NO: 51 or SEQ ID NO: 1264; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 61.

[0020] In some embodiments, an engineered nuclease system is described herein comprising: a nuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 659, 660, 1158, 1159 and 1267-1277; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 726-744, 843-880, 1011-1055 and 1145-1152.

[0021] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 661-678 and 1278-1282; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 745-767 and 881-926.

[0022] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 659; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 699 and 973-974.

[0023] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 696-698; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 700-702 and 957-960.

[0024] In some embodiments, this document describes an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 975-981; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 703-709, 943-944, 951-956, 965-968, and 971-972.

[0025] In some embodiments, engineered guide polynucleotides are single-guide nucleic acids.

[0026] In some embodiments, the engineered guide polynucleotide is a bidirectional guide nucleic acid.

[0027] In some embodiments, the engineered guide polynucleotide is RNA.

[0028] In some embodiments, the endonuclease is not the Cas9 endonuclease.

[0029] In some embodiments, the endonuclease has less than 80% identity with the Cas9 endonuclease.

[0030] In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide.

[0031] In some embodiments, the endonuclease is covalently linked to an engineered guide polynucleotide.

[0032] In some embodiments, the endonuclease is fused with an engineered guide polynucleotide.

[0033] In some embodiments, this document describes methods for modifying target nucleic acid sequences, the methods comprising contacting the target nucleic acid sequence using an engineered nuclease system disclosed herein.

[0034] In some embodiments, the modified target nucleic acid sequence includes binding, cleaving, or cutting the target nucleic acid sequence.

[0035] In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

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

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

[0038] In some embodiments, the modification is ex vivo.

[0039] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1161-1262, 1526-1785, and 2046-2050.

[0040] In some embodiments, this document describes methods for modifying target nucleic acid sequences in mammalian cells, the methods comprising contacting the mammalian cells using an engineered nuclease system disclosed herein.

[0041] In some embodiments, the method further includes selecting cells containing the modification.

[0042] This document provides a method for modifying the hydroxy acid oxidase 1 (HAO1) ​​gene, the method comprising contacting the HAO1 gene with an engineered nuclease system comprising a nuclease containing a sequence having at least 80% sequence identity with SEQ ID NO: 47; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide containing a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1283-1392 and 1502-1509.

[0043] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1283-1392 and 1502-1509.

[0044] In some embodiments, this document describes a method for modifying the ATPase copper transporter β (ATP7B), the method comprising contacting ATP7B with an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence.

[0045] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1056-1081.

[0046] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1206-1231.

[0047] In some embodiments, this document describes a method for modifying adeno-associated virus integration site 1 (AAVS1), the method comprising contacting AAVS1 with an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 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 nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 710-722.

[0048] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 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.

[0049] In some embodiments, the target nucleic acid sequence comprises any one of SEQ ID NO: 59-60, 120-138, 182-200, 243-262, 375-406, 434-446, 473-484, 512-524, 552-564, 1170-1205, 1232, and 1248-1252.

[0050] In some embodiments, this document describes a method for modifying the T-cell receptor α constant (TRAC), the method comprising contacting the TRAC with an engineered nuclease system comprising: a nuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 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 nuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 710-722.

[0051] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 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.

[0052] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 62, 89-100, 151-162, 213-224, 414-420, 454-460, 492-489, 532-538, 1161-1168, and 1200.

[0053] In some embodiments, this document describes a method for modifying an albumin gene, the method comprising contacting the albumin gene with an engineered nuclease system comprising: an endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence.

[0054] In some embodiments, the engineered guide polynucleotide comprises a sequence having any of SEQ ID NO: 1083-1094.

[0055] In some embodiments, the target nucleic acid sequence comprises a sequence having SEQ ID NO: 1233-1244.

[0056] In some embodiments, this document describes a method for modifying the β-2-microglobulin (B2M) gene, the method comprising contacting the B2M gene with an engineered nuclease system comprising: an endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 710-722.

[0057] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 565-578 and 1095.

[0058] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 579-592 and 1245.

[0059] In some embodiments, this document describes a method for modifying the hemoglobin subunit β (HBB) gene, the method comprising contacting the HBB gene with an engineered nuclease system comprising: an endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 710-722.

[0060] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 593-625.

[0061] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 626-658.

[0062] In some embodiments, this document describes a method for modifying a phenylalanine hydroxylase (PAH) gene, the method comprising contacting the PAH gene with an engineered nuclease system comprising: an endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence.

[0063] In some embodiments, the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1113-1122.

[0064] In some embodiments, the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1253-1262.

[0065] In some embodiments, this document describes a method for modifying the ataxia protein 2 (ATXN2) gene, the method comprising contacting the ATXN2 gene with an engineered nuclease system comprising: an endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 47; and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence.

[0066] In some embodiments, the engineered guide polynucleotide comprises the sequence of either SEQ ID NO: 1786-2045 or 2051-2055.

[0067] In some embodiments, the target nucleic acid sequence comprises the sequence of either SEQ ID NO: 1526-1785 or 2046-2050.

[0068] In some embodiments, this document describes cells comprising the engineered nuclease systems disclosed herein.

[0069] 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.

[0070] In some embodiments, the cells are 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 cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0071] In some embodiments, the cells are engineered cells.

[0072] In some embodiments, the cell is a stable cell.

[0073] Further aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious ways without departing from this disclosure. Therefore, the drawings and description are to be regarded in an illustrative rather than restrictive manner. Attached Figure Description

[0074] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments that utilize the principles of this disclosure, and in the accompanying drawings (also referred to herein as “Figure / FIG.”):

[0075] Figure 1 The results of gene editing at the DNA level in TRAC (A1-D2) and AAVS1 (E2-G4) cells in K562 cells, as described in Example 4, are depicted.

[0076] Figure 2 The gene editing results of TRAC at the DNA level in K562 cells, as described in Example 5, are depicted.

[0077] Figure 3A This study describes the results of the first round of gene editing using the guide hAAVS1 C3 at the DNA level to engineer the MG71-2 scaffold.

[0078] Figure 3B Analysis of the second round of engineering of the MG71-2 scaffold using the guide hAAVS1 C3 at the DNA level is described.

[0079] Figure 4 An analysis of gene editing results at the DNA level using the original versus optimized MG71-2 scaffold is presented. Spacers indicated on the Y-axis were tested with 22 nt or 24 nt spacers and the original or shortened MG71-2 scaffold.

[0080] Figures 5A-5B An analysis of gene editing results at the DNA level using the original versus optimized MG71-2 scaffold is presented. Figure 5A The editing results at exon 2 of the human B2M gene using the MG71-2 engineering guide (scaffold 21) are depicted. Figure 5B The editing results at exon 3 of the human HBB gene using the MG71-2 engineering guide (scaffold 21) are depicted.

[0081] Figure 6 A phylogenetic tree of reconstructed ancestral variants of the MG71 family based on multiple sequence alignments was depicted. Ancestor sequences were generated for the nodes of interest (shown with black circles).

[0082] Figure 7 The SeqLogo depicts the protospacer adjacent motif (PAM) identified by MG71-28 obtained from in vitro cleavage assays and NGS sequencing. The MG71 series exhibits a range of PAM specificities, with a preference for A at position 4.

[0083] Figure 8 The SeqLogo depicts the protospacer adjacent motif (PAM) recognized by MG73-6, obtained from in vitro cleavage assays and NGS sequencing. The nuclease MG73-6 is active in vitro in the presence of nnRNCTW PAM.

[0084] Figure 9 The SeqLogo depicts the protospacer adjacent motif (PAM) identified by MG74-8, obtained from in vitro cleavage assays and NGS sequencing. The MG74 family has A / C-rich PAMs.

[0085] Figure 10 The SeqLogo depicts the protospacer adjacent motif (PAM) recognized by MG87-102 obtained from in vitro cleavage assays and NGS sequencing. MG87 family nucleases exhibit the strongest preference for the 5th and 6th bases from the spacer.

[0086] Figure 11 The SeqLogo depicts the protospacer adjacent motif (PAM) identified by MG88-11, obtained from in vitro cleavage assays and NGS sequencing. The MG88 family tends to have purine-rich PAMs at the second to fourth positions.

[0087] Figure 12A-12B The MG71 obtained from in vitro cleavage assays and NGS sequencing was characterized. Figure 12A MG71-42; Figure 12B SeqLogo of the ancestor-recognized protospacer adjacent motif (PAM) of MG71-43. The MG71 ASR protein is active and has a similar or more relaxed PAM to the metagenomic protein MG71-2.

[0088] Figure 13 Gene editing results of AAVS1 at the DNA level (shown as insertion and deletion %) in K562 cells are described.

[0089] Figure 14 A phylogenetic tree of reconstructed ancestral variants of the MG71 family was plotted. The phylogenetic tree was inferred using FastTree from MAFFT multiple sequence alignment. Ancestor sequences were generated for nodes of interest (shown with white circles).

[0090] Figure 15 The predicted alphafold2 structure of MG71-2 was depicted, in which the gRNA and R loop are derived from the crystal structure of SpCas9 bound to substrate DNA and guide (PDB ID: 4UN3). Figure 15 The predicted structure of MG71-2 following the PID mutation is shown. Residues predicted to be within the first interaction shell (5 Å) of the PAM sequence are depicted as bars and labeled.

[0091] Figure 16In vitro PAMs for single-point mutants of MG71-2 and MG71-43 were characterized. The nuclease was expressed in an in vitro transcription / translation reaction and then incubated with a guide 5N plasmid library. The cut plasmids were sequenced and aligned by NGS to generate the PAMs. The first nucleotide "T" at position 0 was masked because it represents a constant side base adjacent to a spacer in the 5N PAM-enriched library (TNNNNN).

[0092] Figure 17 The in vitro PAM reconstructed from the MG71 ancestor was depicted. The nuclease was expressed in an in vitro transcription / translation reaction and then incubated with a guide 5N plasmid library. The cut plasmid was sequenced and aligned by NGS to generate the PAM. The first nucleotide "T" at position 0 was masked because it represents a constant side base adjacent to a spacer in the 5N PAM-enriched library (TNNNNN).

[0093] Figure 18 This study describes the testing of gene-editing activity of MG87-70 in human cells (K562 cells) via nuclear transfection using two doses of the guide. Cells were harvested 72 hours later and gDNA was prepared to evaluate editing via NGS.

[0094] Figure 19 The gene-editing activity of MG87-21 was tested in human cells (K562 cells) via nuclear transfection with 650 pmol gRNA and 500 ng mRNA per well. Cells were harvested 72 hours later and gDNA was prepared to evaluate editing via NGS.

[0095] Figure 20 We characterized the activity of MG71-2 and MG71-43 targeting the intron region between exon 5 and exon 6 of ATP7B in PHH cells. Of the 94 screened guides, 24 were identified by NGS in PHH cells as having an activity >1%.

[0096] Figure 21 We characterized the activity of MG71-2 and MG71-43 targeting the intron region between exon 5 and exon 6 of ATP7B in Hep3B cells. Of the 94 screened guides, 26 were identified by NGS in Hep3B cells as having an activity >1%.

[0097] Figure 22Activity guides targeting the intron 1 region of albumin, specifically MG71-2 and MG71-43 at albumin in PHH cells, were characterized. Of the 19 guides screened, 10 were identified as having >1% activity against one or two nucleases in PHH cells. Additionally, 11 guides were designed specifically for MG71-43; of these, 2 were active in PHH.

[0098] Figures 23A-23C The specificity of MG71-2 and MG71-43 was depicted, demonstrating the high specificity of the MG71 family in mammalian cells. Double-strand break (DSB) formation in cells was assessed by co-nuclear transfection of mRNA, gRNA, and annealed dsODN. dsODN can be incorporated into DSBs and used as a priming site for NGS library preparation, allowing unbiased sampling of all DSBs generated in the cell. The percentage of dsODN reads from the desired target site was plotted for up to three copies of the nuclease MG71-2. Figure 23A For all 8 guides, all reads that passed the analysis criteria originated from the target site. The percentage of dsODN reads from the desired target site was plotted for up to 3 replicas of the nuclease MG71-43. For 5 of the 8 guides, all reads that passed the analysis criteria originated from the target site (…). Figure 23B ). The identified off-target sites for guide g6-MG71-2_AAVS1_H2 are shown relative to the target sequence (top, NNNRCY indicates PAM). Figure 23C The dots indicate matching positions. Boxed bases indicate changes compared to the target. Two off-target sequences are seen in the copy. Figure 23C SEQ ID NO: 2160-2163, 2160, 2163 and 2160-2163 were also disclosed in order of appearance.

[0099] Figures 24A-24B The specificity of MG87-70, which exhibits high specificity in mammalian cells, was depicted. Double-strand break (DSB) formation in cells was assessed by co-nuclear transfection of mRNA, gRNA, and annealed dsODN. dsODN can be incorporated into DSBs and used as a priming site for NGS library preparation, allowing unbiased sampling of all DSBs generated in the cell. The percentage of dsODN reads from the desired target site was plotted for up to three replicas of the nuclease MG87-70. For four of the seven guides, all reads that passed the analysis criteria were derived from the target site (…). Figure 24A The identified off-target sites for guide 87-70_sgRNA_P1B3 are shown relative to the target sequence (top, NNNNGT indicates PAM). Dots indicate matching locations ( Figure 24BThe boxed bases indicate changes compared to the target. Two off-target variations are seen in the copy. Figure 24B SEQ ID NOs 2164-2167, 2164, 2167 and 2164-2167 were also disclosed in order of appearance.

[0100] Figure 25 Pooling screening of MG71 chimeras at PAH p.R408W in engineered immortalized K562 cells was depicted. Each nuclease was delivered as a codon-optimized mRNA along with 10 guide RNAs targeting the SNV of interest. The guides were offset from each other by 1 nt. The cleavage site of each guide was located within 10 nt of the disease mutation. Insertion and deletion efficiencies were evaluated by NGS.

[0101] Figure 26 The activity of engineered human HAO1 MG21-1 guide RNA was described.

[0102] Figure 27 The activity of engineered human AAVS1 MG23-1 guide RNA was described.

[0103] Figure 28 The effect of spacer length on gRNA activity (insertion and deletion %) was depicted. Guided gene editing results from 1–2 different chemically modified backbones with varying spacer lengths from three different type II editing systems (MG23-1, MG21-1, and MG71-2) were analyzed at the DNA level. hAAVS1-23-1-G1-1 (SEQ ID NO: 1486-1493), hTRAC-23-1-H7-1 (SEQ ID NO: 1494-1500), hHAO1-21-1-F1-78 (SEQ ID NO: 1502-1505), hHAO1-21-1-F1-157 (SEQ ID NO: 1506-1509), hTRAC-71-2-C2-12 (SEQ ID NO: 1510-1517), hTRAC-71-2-C2-255 (SEQ ID NO: - 15181525).

[0104] Figure 29 The guide RNA for targeting the human Atxn2 gene with MG21-1 in K562 cells was described. Figure 29Further analysis of the Atxn2 guide using MG21-1 mRNA in K562 cells is presented. MG21-1, along with matching Atxn2 guide RNA (500 ng mRNA / 150 pmol guide), was nuclearly transfected into K562 cells (100,000 cells). Cells were harvested, and genomic DNA was prepared three days post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify individual target sequences for each guide RNA. Amplicon sequencing was performed on an Inmena MiSeq machine, and analysis was conducted using proprietary Python scripts to measure gene editing. The graphs show the guides tested targeting all exons of the Atxn2 region of interest.

[0105] Figure 30 The validation of a subset of the MG21-1 synthesis guide on the Atxn2 gene in human neural progenitor cells was depicted. Figure 30 Further analysis using Atxn2-guided MG21-1 mRNA in human neural progenitor cells (NPCs) is presented. NPC cells were co-transfected with 200 ng mRNA and a chemically synthesized sgRNA targeting Atxn2. Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. Target genomic regions were amplified from the extracted gDNA, and the amplicon was sequenced on the Illumina MiSeq platform. Sequences were analyzed using a proprietary Python script to quantify gene editing efficiency. %OOF indicates out-of-frame mutations.

[0106] Figure 31 A mouse version of the active human synthetic guide was depicted, demonstrating Atxn2 gene-editing activity in mouse neuro-2A cells. Neuro-2A cells were transfected via nuclear transfection using serially diluted buffers of MG21-1 mRNA and a mouse alternative to the Atxn2 guide. The highest concentration of the serially diluted buffer was 500 ng mRNA and 150 pmol guide, followed by a 1:3 serial dilution series. Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. Target genomic regions were amplified from the extracted gDNA using primers designed for NGS-based sequencing. Amplicon sequencing was performed on the Inmena MiSeq platform, and analysis was conducted using proprietary Python scripts to quantify gene-editing efficiency.

[0107] Figure 32The design of an AAV construct carrying an Atxn2 mouse alternative guide RNA was described. The AAV cargo construct was designed to express MG21-1 and the 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 orthogonally upstream of the guide RNA sequence at the 5' end of the cargo. This was followed by a CMV promoter driving the open reading frame of MG21-1. MG21-1 was N-terminally labeled with a V5 epitope. A polyadenylated sequence (SpA) was synthesized following MG21-1.

[0108] Figure 33 An AAV construct carrying MG21-1 was depicted, and a guide targeting mouse Atxn2 was used to demonstrate gene-editing activity in mouse neuro-2A cells. Neuro-2A cells were transfected via liposome transfection using serial dilutions of the AAV cargo plasmid. The highest plasmid concentration in the dilution series was 2 μg, followed by 1:2 serial dilutions. Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. Target genomic regions were amplified from the extracted gDNA using primers designed for NGS-based sequencing. The PCR products were purified, and the amplicon was sequenced. The sequences were analyzed to quantify gene-editing efficiency.

[0109] Figure 34 An AAV construct carrying MG21-1 was depicted, and a guide targeting mouse Atxn2 was used to demonstrate gene-editing activity in mouse Neuro-2A cells. Neuro-2A cells were transfected via liposome transfection using serial dilutions of the AAV cargo plasmid. The highest plasmid concentration in the dilution series was 500 ng, followed by 1:2 serial dilutions. Cells were harvested after three days of culture, and genomic DNA (gDNA) was extracted. Target genomic regions were amplified from the extracted gDNA using primers designed for NGS-based sequencing. The PCR products were purified, and the amplicon was sequenced. The sequences were analyzed using a proprietary Python script to quantify gene-editing efficiency.

[0110] Figure 35An AAV construct carrying MG21-1 was depicted, and a guide targeting mouse Atxn2 demonstrated protein knockdown in mouse neuro-2A cells. Neuro-2A cells were transfected with each of five AAV plasmids (1 µg DNA) containing a different mouse Atxn2 guide using Lipofectamine 2000. Three days post-transfection, cells were lysed and 10 µg was run on a 4–20% Tris-glycine gel. Proteins were transferred to a nitrocellulose membrane, blocked with 5% milk, and then probed with antibodies against the V5 epitope and Atxn2. Blots were repeatedly probed using an actin antibody as a reference control.

[0111] Figure 36 AAV constructs carrying MG21-1 were depicted, and a guide targeting mouse Atxn2 was shown to package the full-length genome into AAV9 via alkaline gel electrophoresis. AAVs were generated in HEK293 cells using a transient triple co-transfection method involving a plasmid encoding an adenovirus accessory protein, a plasmid encoding the AAV replication protein and the AAV9 capsid protein, and a cargo plasmid encoding the guide and MG21-1 side-attached between two AAV2 ITRs. Following transfection, cells were incubated at 37°C for 72 h prior to collection. Crude viral lysates were generated and then treated with cesium chloride density gradient ultracentrifugation before purification. The AAVs were then subjected to three rounds of dialysis before loading onto an alkaline agarose gel to visualize the size and integrity of the vector genome.

[0112] Figure 37 The in vivo study design, experimental procedures, and sample preparation are described. AAV9 was injected into both the olfactory bulb and the M1 region of the motor cortex of 6–8-week-old C57BL / 6J mice using a custom-built stereotactic delivery system based on a RWD automated stereotactic instrument. M1 injections consisted of bilateral infusions of 750 nL AAV9 at coordinates +1.00 AP, + / -1.50 ML, and -1.50 DV relative to the anterior fontanelle at three target depths: +1.00 AP, + / -0.70 ML, and -2.5 / -2.0 / -1.5 DV. Olfactory bulb injections consisted of bilateral infusions of 250 nL AAV9 at three target depths at coordinates +4.00 AP, + / -0.70 ML, and -2.5 / -2.0 / -1.5 DV relative to the anterior fontanelle at three target depths: +4.00 AP, + / -0.70 ML, and -2.5 / -2.0 / -1.5 DV. A 5-minute retention time was added post-injection to enhance AAV absorption into the tissues. Animals were sutured and allowed to recover until transfer back to their cages. Two weeks after injection, the animals were euthanized by deep inhalation of isoflurane. The whole brain was freshly dissected, and the olfactory bulb and M1 region were further dissected and processed for downstream nucleic acid extraction. Genomic DNA and mRNA were extracted from the fresh tissue using a DNA and RNA separation kit.

[0113] Figure 38 Tissue titer measurements were depicted, confirming that MG21-1 AAV effectively transduced the olfactory bulb and motor cortex of mice. Internally prepared AAV9 was injected into both the olfactory bulb and the M1 region of the motor cortex of 6–8-week-old C57BL / 6J mice using a custom-built stereotactic delivery system. Two weeks post-injection, animals were euthanized by deep inhalation of isoflurane. The whole brain was freshly dissected, and the olfactory bulb and M1 region were further dissected and processed for downstream nucleic acid extraction. To determine the tissue titer of the injected AAV, 5 ng gDNA was added to a ddPCR reaction containing a custom probe targeting the central region of the MG21-1 sequence in multiplex with a pre-designed assay targeting the CyC1 gene. The MG21-1 copy number was normalized to a CyC1 reference copy number to produce a normalized MG21-1 copy / cell value. All individual data points are shown with error bars representing the mean plus or minus the standard error of the mean.

[0114] Figure 39 This study depicts the detection of MG21-1 transcripts in the olfactory bulb and motor cortex following AAV9 injection. Internally prepared AAV9 was injected into both the olfactory bulb and the M1 region of the motor cortex of 6–8-week-old C57BL / 6J mice using a custom-built stereotactic delivery system. Animals were euthanized two weeks post-injection by deep inhalation of isoflurane. The whole brain was freshly dissected, with further dissection and processing of the olfactory bulb and M1 region for downstream nucleic acid extraction. mRNA was extracted from the fresh tissue and then used to generate cDNA via reverse transcription. To quantify MG21-1 expression, 5 ng 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 also multiplexed with an assay targeting the Cyc1 gene. Data were plotted in GraphPad Prism 10. MG21-1 copies were normalized to a Cyc1 reference copy to produce a normalized MG21-1 copy / Cyc1 copy value. All individual data points are shown in error bars, representing the standard error of the mean plus or minus the mean.

[0115] Figure 40This study demonstrated that MG21-1, packaged in AAV9, effectively knocked down Atxn2 mRNA in both the olfactory bulb and the motor cortex. Internally prepared AAV9 was injected into both the olfactory bulb and the M1 region of the motor cortex of 6–8-week-old C57BL / 6J mice using a custom-built stereotactic delivery system. Two weeks post-injection, animals were euthanized by deep inhalation of isoflurane. The whole brain was freshly dissected, with further dissection and processing of the olfactory bulb and M1 region for downstream nucleic acid extraction. mRNA was extracted from the fresh tissue and then used to generate cDNA via reverse transcription. To quantify Atxn2 expression, 5 ng of cDNA mRNA equivalent was loaded into a ddPCR reaction containing a pre-prepared Atxn2 expression quantification assay. This assay was also multiplexed with a pre-prepared assay targeting the Cyc1 gene. Data were plotted in GraphPad Prism 10. Atxn2 mRNA copies were normalized relative to a Cyc1 reference copy to produce a normalized Atxn2 copy / Cyc1 copy value. All individual data points are shown in error bars, representing the standard error of the mean plus or minus the mean.

[0116] Figure 41 A plot depicting insertions and deletions in the mouse Atxn2 gene detectable in the mouse olfactory bulb and motor cortex is presented. All individual data points are shown with error bars representing the mean plus or minus the standard error of the mean.

[0117] Figure 42 A plot depicting the quantification of Atxn2 protein expression in olfactory bulb samples injected with AAV9 was created. All individual data points are shown with error bars representing the mean plus or minus the standard error of the mean.

[0118] Figure 43 Data from stereotactic injection of AAV from MG21-1, which encodes Atxn2, are depicted, and target protein knockdown is shown by imaging. Scale bars represent 70 µm.

[0119] Figure 44 Stereotactic injection of AAV from the MG21-1_P1F12-encoding guide was depicted by imaging data showing Atxn2 protein knockdown. Scale bar indicates 70 µm.

[0120] Figure 45 A schematic overview of the workflow is depicted. A single mismatch (1MM target) is generated across each location of the spacer and PAM, and used to create members of a dual-target library. The library is sequenced and cloned into a lentiviral plasmid to generate a plasmid library from which lentiviral libraries are derived. K562 cells are infected with the dual-target lentibrary, and after antibiotic selection, cells are edited via nuclear transfection and gDNA is collected. Data are analyzed after NGS sequencing.

[0121] Figure 46A and 46B A bar graph was plotted to show the GC content and insertion and deletion percentages of the library's sgRNAs. The GC content of the library, as designed by the nuclease, was also plotted using nucleases. Figure 46A ) and percentage of insertions and missing values ​​( Figure 46B ) range.

[0122] Figure 47 A schematic overview of the reference structures of members of a dual-target oligonucleotide library is provided.

[0123] Figure 48 A diagram illustrating the edit controls is shown. Control oligonucleotides were generated to simulate four possible edit events: cleavage at the target site, cleavage at the off-target site, cleavage at both targets, and no cleavage. These control dual-target sequences have identical on-target and off-target spacers, where the cleavage event is regulated by their active or inactive PAM sequence. To generate inactive PAMs, bases in the active PAM common sequence were substituted with alternative bases not recognized by the Cas system. Edits at the left or right ends of the dual-target controls following nuclear transfection using a guide across MG71-2 and MG21-1 are plotted.

[0124] Figure 49 A nuclease-specific single mismatch tolerance map was generated. For each library member, the percentage of mismatched targets and their corresponding percentages of original insertions and deletions at the target were calculated. The percentages of original insertions and deletions were normalized across each position by obtaining the ratio of observed close:edits at the target (referred to as mismatch tolerance) for each wizard. A nuclease-specific single mismatch tolerance map was generated by plotting the mean and 95% confidence intervals of these single mismatch tolerances for all wizards of a given nuclease at each position.

[0125] Figure 50 A plot illustrating the individual mismatch tolerance of the 22-nucleotide spacer of MG71-2 was created. Dual-target library members were designed using 25 wizards targeting human B2M, TRAC, and AAVS1 loci, with off-target sequences differing by one mismatch between the off-target and target sequences. The percentage of mismatched targets relative to the original insertions and deletions at the target library members was normalized by taking the off-target:target editing ratio (referred to as mismatch tolerance) observed at each wizard position, and nuclease-specific maps were generated by plotting the mean and 95% confidence intervals of individual mismatch tolerances at each position.

[0126] Figure 51A plot illustrating the individual mismatch tolerance of the 24-nucleotide spacer of MG71-2 was created. Dual-target library members were designed using seven wizards targeting the human HBB locus, where off-target sequences differed by one mismatch between the off-target and on-target sequences. The percentage of mismatched targets relative to the original insertions and deletions at the target library members was normalized by taking the off-target:on-target editing ratio (referred to as mismatch tolerance) observed at each wizard position, and a nuclease-specific map was generated by plotting the mean and 95% confidence intervals of individual mismatch tolerances at each position.

[0127] Figure 52 Overlapping plots illustrating the individual mismatch tolerance of MG71-2 with 22 and 24 nucleotide spacers are depicted. Dual-target library members were designed using 25 guides of 22 nt length targeting the human B2M, TRAC, and AAVS1 loci and 7 guides of 24 nt length targeting the human HBB locus, where off-target sequences differed by one mismatch between the off-target and on-target sequences. The percentage of mismatched targets relative to the original insertions and deletions at the target library members was normalized by taking the off-target:on-target editing ratio (referred to as mismatch tolerance) observed at each guide position, and nuclease-specific maps were generated by plotting the mean and 95% confidence intervals of individual mismatch tolerances by position. The two plots were overlaid for visualization.

[0128] Figure 53 A plot illustrating the individual mismatch tolerance of MG21-1 was created. Dual-target library members were designed using 21 wizards targeting human PDCD1, HAO1, IL17, and TRAC loci, where off-target sequences differed by one mismatch from the target sequence. The percentage of mismatched targets relative to the original insertions and deletions at the target library members was normalized by taking the off-target:target editing ratio (referred to as mismatch tolerance) observed at each wizard position, and a nuclease-specific map was generated by plotting the mean and 95% confidence intervals of individual mismatch tolerances at each position.

[0129] Figure 54 A graph illustrating the PAM preferences of MG71-2 is depicted. The PAM preference score of MG71-2 is calculated by converting the mismatch tolerance score and its corresponding off-target PAM into an information-based representation matrix using a tagger.

[0130] Figure 55 A graph illustrating the PAM preferences of MG21-1 is depicted. The PAM preference score of MG21-1 is calculated by converting the mismatch tolerance score and its corresponding off-target PAM into an information-based representation matrix using a tagger.

[0131] Figure 56A heatmap of protospacer base preferences for MG71-2 was created. The heatmap was constructed to show the protospacer base preferences for different nucleases at each mismatch site. The heatmap was generated by grouping data points by nuclease, mismatch site, and mismatched protospacer base, and then calculating the average off-target-on-target ratio and 95% confidence interval for each group.

[0132] Figure 57 A heatmap of MG21-1 protospacer base preference was plotted. The heatmap was created to show the protospacer base preference for different nucleases at each mismatch site. The heatmap was generated by grouping data points by nuclease, mismatch site, and mismatched protospacer base, and then calculating the average off-target-on-target ratio and 95% confidence interval for each group.

[0133] Brief description of sequence lists

[0134] The sequence listing submitted herein provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions, and systems according to this disclosure. An exemplary description of the sequences therein is given below.

[0135] MG21

[0136] SEQ ID NO: 47 shows the full-length peptide sequence of the MG21 nuclease.

[0137] SEQ ID NO: 1137-1144 shows the nucleotide sequence of sgRNA engineered to function in conjunction with the MG21 nuclease.

[0138] MG23

[0139] SEQ ID NO: 48 shows the full-length peptide sequence of the MG23 nuclease.

[0140] SEQ ID NO: 1153-1156 shows the nucleotide sequences of sgRNA engineered to function in conjunction with the MG23 nuclease.

[0141] MG71

[0142] SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010 and 1263 show the full-length peptide sequences of the MG71 nuclease.

[0143] SEQ ID NO: 53-54 shows the peptide sequence of the PAM interaction domain of the MG71 nuclease.

[0144] nnRMYnn, nnnACTnn, nNNRMT, nRNACT, nNNACT, nRNRCT, nNNRHY, nNNRMT, nNNRCY, nNNRYY, nNNRMT, nNNRCY, nNWRAT, nNNRMT, nNNRHT, nNNRHY, nNNRHY, nNNRHY, nNNRHY, nRNRYY, nRNRCN, nNNRYY, nRNRNT, nNNAWT, nNRAMW, nNARMY, nNNRCT, nNNRYT, nNARYY, nNNRCY and nNNRYY are PAM sequences compatible with MG71 nuclease.

[0145] SEQ ID NO: 710-722 shows the nucleotide sequence of MG71 tracrRNA.

[0146] SEQ ID NO: 779-791 shows the nucleotide sequence of the MG71 CRISPR repeat sequence.

[0147] 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 in conjunction with the MG71 nuclease. In SEQ ID NOs: 2433-2434, "n" can be any nucleotide.

[0148] MG73

[0149] SEQ ID NO: 51 and 1264 show the full-length peptide sequences of the MG73 nuclease.

[0150] SEQ ID NO: 55 shows the peptide sequence of the PAM interaction domain of the MG73 nuclease.

[0151] nnRnTTnn is a PAM sequence compatible with MG73 nuclease.

[0152] MG89

[0153] SEQ ID NO: 52 shows the full-length peptide sequence of the MG89 nuclease.

[0154] SEQ ID NO: 56 shows the peptide sequence of the PAM interaction domain of the MG89 nuclease.

[0155] nnnnCC is a PAM sequence compatible with MG89 nuclease.

[0156] MG87

[0157] SEQ ID NO: 659-660, 1158-1159 and 1267-1277 show the full-length peptide sequence of the MG87 nuclease.

[0158] SEQ ID NO: 726-744 shows the nucleotide sequence of MG87 tracrRNA.

[0159] SEQ ID NO: 795-813 shows the nucleotide sequence of the MG87 CRISPR repeat sequence.

[0160] SEQ ID NO: 843-880 and 1145-1152 show the nucleotide sequences of sgRNAs engineered to function in conjunction with the MG87 nuclease.

[0161] MG88

[0162] SEQ ID NO: 661-678 and 1278-1282 show the full-length peptide sequences of the MG88 nuclease.

[0163] SEQ ID NO: 745-767 shows the nucleotide sequence of MG88 tracrRNA.

[0164] SEQ ID NO: 814-836 shows the nucleotide sequence of the MG88 CRISPR repeat sequence.

[0165] SEQ ID NO: 881-926 shows the nucleotide sequence of sgRNA engineered to function in conjunction with the MG88 nuclease.

[0166] MG17

[0167] SEQ ID NO: 695 shows the full-length peptide sequence of the MG17 nuclease.

[0168] SEQ ID NO: 699 shows the nucleotide sequence of MG17 tracrRNA.

[0169] SEQ ID NO: 768 shows the nucleotide sequence of the MG17 CRISPR repeat sequence.

[0170] SEQ ID NO: 973-974 shows the nucleotide sequence of sgRNA engineered to function in conjunction with the MG17 nuclease.

[0171] MG18

[0172] SEQ ID NO: 696-698 shows the full-length peptide sequence of the MG18 nuclease.

[0173] SEQ ID NO: 700-702 shows the nucleotide sequence of MG18 tracrRNA.

[0174] SEQ ID NO: 769-771 shows the nucleotide sequence of the MG18 CRISPR repeat sequence.

[0175] SEQ ID NO: 957-960 shows the nucleotide sequence of sgRNA engineered to function in conjunction with the MG18 nuclease.

[0176] MG46

[0177] SEQ ID NO: 975-981 shows the full-length peptide sequence of the MG46 nuclease.

[0178] SEQ ID NO: 703-709 shows the nucleotide sequence of MG46 tracrRNA.

[0179] SEQ ID NO: 772-778 shows the nucleotide sequence of the MG46 CRISPR repeat sequence.

[0180] SEQ ID NO: 943-944, 951-956, 965-968 and 971-972 show the nucleotide sequences of sgRNAs engineered to function in conjunction with the MG46 nuclease.

[0181] MG74

[0182] SEQ ID NO: 1265-1266 shows the full-length peptide sequence of the MG74 nuclease.

[0183] Targeting HAO1 with MG21-1

[0184] SEQ ID NO: 1283-1392 and 1502-1509 show nucleotide sequences of sgRNA engineered to work with the MG21-1 nuclease to target the human HAO1 gene.

[0185] MG71-2 AAVS1 Target

[0186] SEQ ID NO: 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 sgRNA engineered to work with the MG71-2 nuclease to target the AAVS1 gene.

[0187] SEQ ID NO: 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 the AAVS1 target site.

[0188] MG71-2 TRAC Targeted

[0189] SEQ ID NO: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531 and 1096-1097 show nucleotide sequences of sgRNA engineered to work with the MG71-2 nuclease to target TRAC.

[0190] SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538 and 1246-1247 show the DNA sequences of the TRAC target sites.

[0191] MG71-2 B2M Targeted

[0192] SEQ ID NO: 565-578 and 1095 show nucleotide sequences engineered to work with the MG71-2 nuclease to target the human B2M gene sgRNA.

[0193] SEQ ID NO: 579-592 and 1245 show the DNA sequences of the human B2M target site.

[0194] MG71-2 HBB Targeted

[0195] SEQ ID NO: 593-625 shows the nucleotide sequence of sgRNA engineered to work with the MG71-2 nuclease to target the human HBB gene.

[0196] SEQ ID NO: 626-658 shows the DNA sequence of the human HBB target site.

[0197] MG71-2 albumin-targeted

[0198] SEQ ID NO: 1083-1094 shows the nucleotide sequence of sgRNA engineered to work with the MG71-2 nuclease to target the human albumin gene.

[0199] SEQ ID NO: 1233-1244 shows the DNA sequence of the human albumin target site.

[0200] MG71-2 ATP7B Target

[0201] SEQ ID NO: 1056-1081 shows the nucleotide sequence of sgRNA engineered to work with the MG71-2 nuclease to target human ATP7B.

[0202] SEQ ID NO: 1206-1231 shows the DNA sequence of the human ATP7B target site.

[0203] MG71-2 PAH Target

[0204] SEQ ID NO: 1113-1122 shows the nucleotide sequence of sgRNA engineered to work with the MG71-2 nuclease to target the human PAH gene.

[0205] SEQ ID NO: 1253-1262 shows the DNA sequence of the human PAH target site.

[0206] MG73-1 TRAC Targeted

[0207] SEQ ID NO: 61 shows the nucleotide sequence of the sgRNA engineered to work with the MG73-1 nuclease to target TRAC.

[0208] SEQ ID NO: 62 shows the DNA sequence of the TRAC target site.

[0209] MG89-2 TRAC Targeted

[0210] SEQ ID NO: 63-69 and 263-302 show nucleotide sequences of sgRNA engineered to work with the MG89-2 nuclease to target TRAC.

[0211] SEQ ID NO: 70-76 and 303-342 show the DNA sequences of the TRAC target sites.

[0212] MG87 TRAC Target

[0213] SEQ ID NO: 1011-1019 and 1050 show nucleotide sequences of sgRNA engineered to work with the MG87 nuclease to target TRAC.

[0214] SEQ ID NO: 1161-1168 and 1200 show the DNA sequences of the TRAC target sites.

[0215] MG87 AAVS1 Target

[0216] SEQ ID NO: 1020-1049 and 1051-1055 show the nucleotide sequences of sgRNA engineered to work with the MG87 nuclease to target the AAVS1 gene.

[0217] SEQ ID NO: 1169-1205 shows the DNA sequence of the AAVS1 target site.

[0218] MG21-1 ATXN2 Target

[0219] SEQ ID NO: 1786-2045 shows the nucleotide sequence of sgRNA engineered to work with the MG21-1 nuclease to target the human ATXN2 gene.

[0220] SEQ ID NO: 2051-2055 shows the nucleotide sequences of sgRNA engineered to work with the MG21-1 nuclease to target the mouse ATXN2 gene.

[0221] SEQ ID NO: 1526-1785 shows the DNA sequence of the human ATXN2 target site.

[0222] SEQ ID NO: 2046-2050 shows the DNA sequence of the mouse ATXN2 target site.

[0223] MG23-1 AAVS1 Target

[0224] SEQ ID NO: 1393-1493 shows the nucleotide sequence of sgRNA engineered to work with the MG23-1 nuclease to target the AAVS1 gene.

[0225] MG23-1 TRAC Targeted

[0226] SEQ ID NO: 1494-1501 and 1510-1525 show nucleotide sequences of sgRNA engineered to work with the MG23-1 nuclease to target TRAC.

[0227] AAV cargo particles

[0228] SEQ ID NO: 2056-2060 shows the nucleic acid sequences of the AAV cargo particles. Detailed Implementation

[0229] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various changes, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0230] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first value in a series of two or more values, the terms "at least," "greater than," or "greater than or equal to" apply to each value in the series. 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.

[0231] Whenever the terms "not exceeding," "less than," or "less than or equal to" precede the first value in a series of two or more values, the terms "not exceeding," "less than," or "less than or equal to" apply to each value in the series. 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.

[0232] Unless otherwise indicated, the practice of some of the methods disclosed herein employs techniques from immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA.

[0233] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Furthermore, where the terms “including,” “include,” “having,” “has,” “with,” or variations thereof are used in the Detailed Description and / or Claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0234] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0235] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Contemplated nucleotides include both naturally occurring and synthetic nucleotides. A nucleotide is a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates, adenosine triphosphates (ATP), uridine triphosphates (UTP), cytosine triphosphates (CTP), guanosine triphosphates (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled, such as using portions containing an optically detectable portion (e.g., a fluorophore) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, 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), Waterfall Blue, Oregon Green, Texas Red, Cyanide, and 5-(2'-aminoethyl)aminonaphthalene-1-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, all available from Perkin Elmer, FosterCity, Calif.; and FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink, all available from Amersham, Arlington Heights, IL. Cy5-dUTP; luciferin-15-dATP, luciferin-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, luciferin-12-ddUTP, luciferin-12-UTP, and luciferin-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and luciferin-15-2'-dATP, available from Molecular Probes, Eugene, Chromosome-tagged nucleotides obtained from Oreg, including BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Waterfall Blue-7-UTP, Waterfall 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. 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).

[0236] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or their analogues, in single-stranded, double-stranded, or multi-stranded form. Contemplated polynucleotides include genes or segments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene segments, multiple loci (locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (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 polynucleotides, when T is mentioned, T refers to U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in cell-free environments. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbone, sugars, or nucleobases). Modifications to the nucleotide structure are conferred, if present, before or after polymer assembly. Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acids, heteronucleotides, morpholinonucleotides, locked nucleic acids, glycerol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-denitro-GTP, fluorophores (e.g., sugar-linked rhodamine or fluorescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, brassinoside, and woyoside. The sequence of a nucleotide can be interrupted by non-nucleotide components.

[0237] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers containing at least two amino acid residues linked by peptide bonds. This terminology does not indicate a specific length of the polymer, nor is it intended to imply or distinguish whether a peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may contain non-amino acid compounds. These terms include amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structures (e.g., domains). These terms also cover modified amino acid polymers; for example, through disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with labeled components. As used herein, the term “amino acid” refers to both natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include natural amino acids that have been chemically modified to include groups or chemical moieties on the amino acid that are not naturally occurring. The term “amino acid” includes both D-amino acids and L-amino acids.

[0238] As used herein, “operationally linked,” “operationally connected,” “operationally connected,” or their syntactic equivalents refer to the arrangement of genetic elements, such as promoters, enhancers, polyadenylated sequences, etc., in which an operation (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element can be, but does not have to be, the same type of operation as that of the first genetic element. For example, if movement of the first element results in activation of the second element, the two genetic elements are operably linked. For example, if a regulatory element contributes to initiating transcription of a coding sequence, a regulatory element containing promoter and / or enhancer sequences can be operably linked to a coding region. Insertion residues may exist between the regulatory element and the coding region as long as this functional relationship is maintained.

[0239] A “functional segment” of a DNA or protein sequence refers to a segment that retains biological activity (functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to influence expression in a manner attributable to the full-length sequence.

[0240] The terms “engineered,” “synthetic,” and “artificial” are used interchangeably herein to refer to objects modified through human intervention. For example, these terms refer to non-naturally occurring polynucleotides or peptides. Engineered peptides have, but do not require, low sequence identity with naturally occurring human proteins (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). For example, the VPR and VP64 domains are synthetic trans-activation domains. Non-limiting examples include: nucleic acids being modified by altering their sequence to a sequence not found in nature; nucleic acids being modified by ligating them to nucleic acids that do not associate with them in nature, such that the ligation product has a function not present in the original nucleic acid; engineered nucleic acids being synthesized in vitro with sequences not found in nature; proteins being modified by altering the amino acid sequence of a protein to a sequence not found in nature; and engineered proteins acquiring new functions or properties. An “engineered” system contains at least one engineered component.

[0241] The term "tracrRNA" or "tracr sequence" refers to trans-activated CRISPR RNA. tracrRNA interacts with CRISPR(cr)RNA to form a guide RNA (e.g., guide RNA or gRNA) that can hybridize with the target nucleic acid and thereby guide the associated nuclease to the target nucleic acid.

[0242] As used herein, “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that can hybridize with a target nucleic acid and thereby guide 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 may include crRNA or tracrRNA or a combination of both. The term guide nucleic acid encompasses engineered guide nucleic acids and programmable guide nucleic acids for specific binding to a 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 a double-stranded target polynucleotide that is complementary to the complementary strand and therefore not complementary to the guide nucleic acid is called the non-complementary 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.” Unless otherwise stated, the term “guide nucleic acid” is inclusive and refers to both single and double guide nucleic acids. A guide nucleic acid may contain a segment referred to as a “nucleic acid targeting segment,” “nucleic acid targeting sequence,” or “spacer.” Nucleic acid targeting regions may include sub-regions, which are referred to as “protein-binding regions” or “protein-binding sequences” or “Cas protein-binding regions”.

[0243] As used herein, the term "complex" refers to the connection of at least two components. The two components may each retain their properties / activities prior to the formation of the complex or acquire properties as a result of the complex's formation. Connections include, but are not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonds), the use of linkers, fusion, or any other suitable method. The components of the envisioned complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex may contain an endonuclease and a guide polynucleotide.

[0244] In the context of two or more nucleic acid or polypeptide sequences, the terms “sequence identity” or “percentage identity” refer to two (e.g., in paired alignments) or more (e.g., in multiple sequence alignments) sequences that are identical or have the same specific percentage of amino acid residues or nucleotides when compared and aligned within a local or global comparison window to obtain maximum correspondence, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for peptide sequences include, for example: BLASTP for peptide sequences longer than 30 residues, using a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix with a gap penalty of 11 for presence, 1 for expansion, and conditional composition of the scoring matrix; BLASTP for sequences shorter than 30 residues, using a word length (W) of 2, an expected value (E) of 1000000, and a PAM30 scoring matrix with a gap penalty of 9 for gap opening and 1 for expansion (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW with parameters for the Smith-Waltman homology search algorithm using a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default parameters; MAFFT with a retree of 2 and a maximum iteration of 1000; and Novafold with default parameters.

[0245] This disclosure includes variants of any of the enzymes described herein that have one or more conserved amino acid substitutions. Such conserved substitutions can occur in the amino acid sequence of a polypeptide without disrupting the polypeptide's three-dimensional structure or function. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R-chain lengths into each other. Alternatively or additionally, conserved substitutions can be identified by comparing aligned sequences of homologous proteins from different species, by locating mutated amino acid residues between species (e.g., non-conserved residues) without altering the fundamental function of the encoded protein. Such conserved substitution variants may include variants 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 with any 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 conserved substitution variants are functional variants. Such functional variants may encompass sequences with substitutions such that the activity of key active site residues of the endonuclease is not compromised. In some embodiments, any functional variant of the protein described herein lacks substitution for at least one of the residues predicted to be required. In some embodiments, any functional variant of the protein described herein lacks substitution for all of the residues predicted to be required.

[0246] Conserved substitutions of functionally similar amino acids can be obtained from various references (see, for example, Creighton, *Proteins: Structures and Molecular Properties* (WH Freeman & Co.); 2nd ed. (December 1993)). The following eight groups each contain amino acids that are conserved in their substitutions:

[0247] 1) Alanine (A), glycine (G);

[0248] 2) Aspartic acid (D), glutamic acid (E);

[0249] 3) Asparagine (N), glutamine (Q);

[0250] 4) Arginine (R), Lysine (K);

[0251] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0252] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0253] 7) Serine (S), threonine (T); and

[0254] 8) Cysteine ​​(C), Methionine (M)

[0255] Overview

[0256] The discovery of novel Cas enzymes with unique functions and structures may offer the potential to further disrupt DNA editing technologies, thereby improving speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly spaced short palindromic repeat (CRISPR) systems in microorganisms and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a large number of microbial species may not be easily cultured under laboratory conditions. Metagenomic sequencing of natural environmental niches representing a large number of microbial species could offer the potential to significantly increase the number of known novel CRISPR / Cas systems and accelerate the discovery of new oligonucleotide editing functions. A recent, fruitful example of this approach is evidenced by the discovery of the CasX / CasY CRISPR system in 2016 through metagenomic analysis of a natural microbial community.

[0257] The CRISPR / Cas system is an RNA-guided nuclease complex that has been described as acting as an adaptive immune system in microorganisms. In its natural environment, the CRISPR / Cas system occurs within CRISPR (clustered, regularly spaced short palindromic repeats) operons or loci, typically comprising two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences, encoding RNA-based targeting elements; and (ii) an ORF encoding Cas, which encodes a nuclease polypeptide guided by the RNA-based targeting element along with an accessory protein / enzyme. Efficient nuclease targeting of a specific target nucleic acid sequence typically requires both: (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer neighbor motif (PAM) sequence (PAMs are often sequences not commonly represented within the host genome) in the vicinity of the defined target seed. Based on the system's exact function and organization, CRISPR-Cas systems are generally classified into 2 classes, 5 types, and 16 subtypes based on shared functional characteristics and evolutionary similarities.

[0258] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include types I, III, and IV.

[0259] Type I CRISPR-Cas systems are considered moderately complex in terms of composition. In a type I CRISPR-Cas system, an array of RNA-targeting elements is transcribed into long precursor crRNA (pre-crRNA), which is processed at repeating elements to release short mature crRNA. This short mature crRNA, followed by a suitable short concordant sequence called a protospacer neighbor motif (PAM), directs the nuclease complex to the nucleic acid target. This processing is carried out via an endonuclease subunit (Cas6) of a large endonuclease complex called a cascade, which also contains the crRNA-guided nuclease complex nuclease (Cas3) protein component. Cas1 nucleases primarily function as DNA nucleases.

[0260] The type III CRISPR system is characterized by the presence of a central nuclease called Cas10 and a repeat-associated mystery protein (RAMP) containing either a Csm or Cmr protein subunit. As in the type I system, mature crRNA is processed from pre-crRNA using a Cas6-like enzyme. Unlike the type I and type II systems, the type III system appears to target and cleave the DNA-RNA duplex (such as the DNA strand used as a template for RNA polymerase).

[0261] Type IV CRISPR-Cas systems have an effector complex consisting of two genes of RAMP proteins from the highly reduced large subunit nuclease (csf1), Cas5 (csf3), and Cas7 (csf2) groups, as well as genes of the predicted small subunit in some cases; such systems are typically found on endogenous plasmids.

[0262] Class II CRISPR-Cas systems typically have single-peptide multi-domain nuclease effectors and include types II, V, and VI.

[0263] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, processing a CRISPR array into mature crRNA does not require the presence of a specific endonuclease subunit; instead, it requires a small trans-coding crRNA (tracrRNA) whose region is complementary to the array repetitive sequence. The tracrRNA interacts with its corresponding effector nuclease (Cas9) and the repetitive sequence to form a precursor dsRNA structure. This precursor dsRNA structure is cleaved by endogenous RNase III to produce a mature Cas9 enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are called DNA nucleases. The Cas9 effector has a characteristic structure consisting of RuvC-like endonuclease domains that employ RNase H folds, in which an unrelated HNH nuclease domain is inserted. The RuvC-like domain is responsible for cleaving the target (e.g., crRNA complementary) DNA strand, while the HNH domain is responsible for cleaving the replaced DNA strand.

[0264] Type V CRISPR-Cas systems are characterized by a nuclease effector (Cas12) structure similar to that of type II / Cas9, which contains a RuvC-like domain. Similar to type II, most (but not all) type V CRISPR systems use tracrRNA to process precursor crRNA into mature crRNA; however, unlike type II systems which require RNase III to cleave the precursor crRNA into multiple crRNAs, type V systems are able to use the effector nuclease itself (Cas12) to cleave the precursor crRNA. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are again referred to as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to possess robust single-stranded nonspecific deoxyribonuclease activity activated by the directed cleavage of the first crRNA from the double-stranded target sequence.

[0265] The type VI CRISPR-Cas system is unique because it appears to be the only class to date that is classified as an RNA-guided RNA endonuclease. The single polypeptide effector of the type VI system (Cas13) contains two HEPN ribonuclease domains, rather than a RuvC-like domain. Unlike both type II and type V systems, the type VI system also does not appear to require tracrRNA to process the precursor crRNA into crRNA. However, similar to the type V system, some type VI systems (e.g., C2C2) appear to possess robust single-stranded non-specific nuclease (ribonuclease) activity activated by the first crRNA-directed cleavage of the target RNA.

[0266] Due to its simpler architecture, the Class 2 CRISPR-Cas has been most widely used in engineering and development as a designer nuclease / genome editing application.

[0267] This system is used for one of the early adaptations in vitro using (i) recombinant expression and purification of full-length Cas9 (e.g., class 2, type II Cas enzyme) isolated from Streptococcus pyogenes SF370; (ii) purified mature approximately 42 nt crRNA carrying an approximately 20 nt 5' sequence complementary to the target DNA sequence to be cleaved, followed by a 3' tracr binding sequence (the entire crRNA was transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence); (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template carrying a T7 promoter sequence; and (iv) Mg 2+ Later improved engineered systems involved (ii) crRNA binding to the 5' end of (iii) via a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) capable of guiding Cas9 to the target itself.

[0268] Such engineered systems can be adapted for mammalian cells by providing DNA vectors encoding the following: (i) an ORF encoding a codon-optimized Cas9 (e.g., type II Cas enzyme) with a suitable mammalian promoter having 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 (with a 5' sequence starting with G followed by a 20 nt complementary targeting nucleic acid sequence that binds to a 3' tracr binding sequence, a linker, and a tracrRNA sequence) with a suitable polymerase III promoter (e.g., U6 promoter).

[0269] MG enzyme

[0270] In some embodiments, this document describes engineered nuclease systems comprising a nuclease and an engineered guide polynucleotide. In some embodiments, the nuclease is a type II nuclease. In some embodiments, the nuclease comprises a RuvC_III domain. In some embodiments, the nuclease comprises an HNH domain. In some embodiments, the nuclease is a double-stranded nuclease. In some embodiments, the nuclease undergoes catalytic death. In some embodiments, the nuclease is a double-stranded nuclease. In some embodiments, the nuclease is modified. In some embodiments, the nuclease is modified to produce a nuclease with nicking enzyme activity. In some embodiments, the modified nuclease is a site-directed nicking enzyme.

[0271] In some embodiments, the endonuclease is MG21 endonuclease. In some embodiments, the endonuclease is MG23 endonuclease. In some embodiments, the endonuclease is MG71 endonuclease. In some embodiments, the endonuclease is MG73 endonuclease. In some embodiments, the endonuclease is MG74 endonuclease. In some embodiments, the endonuclease is MG89 endonuclease. In some embodiments, the endonuclease is MG87 endonuclease. In some embodiments, the endonuclease is MG88 endonuclease. In some embodiments, the endonuclease is MG17 endonuclease. In some embodiments, the endonuclease is MG18 endonuclease. In some embodiments, the endonuclease is MG46 endonuclease.

[0272] In some embodiments, engineered nuclease systems are discovered through metagenomic sequencing. In some embodiments, the metagenomic sequencing is performed on samples collected from various environments. In some embodiments, the environment is a human microbiome, an animal microbiome, a high-temperature environment, a low-temperature environment, or sediment.

[0273] In some embodiments, the endonuclease is the 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 with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 47. In some embodiments, the endonuclease comprises a sequence having 100% identity with SEQ ID NO: 47.

[0274] In some embodiments, the endonuclease is an 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 with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 48. In some embodiments, the endonuclease comprises a sequence having 100% identity with SEQ ID NO: 48.

[0275] In some embodiments, the endonuclease is an MG71 endonuclease (i.e., SEQ ID NO: 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 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 with any of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263.In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263. In some embodiments, the endonuclease comprises a sequence having 100% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263.

[0276] In some embodiments, the endonuclease is the 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 with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some embodiments, the endonuclease comprises a sequence having 100% identity with SEQ ID NO: 51 or SEQ ID NO: 1264.

[0277] In some embodiments, the endonuclease is the 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 with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 52. In some embodiments, the endonuclease comprises a sequence having 100% identity with SEQ ID NO: 52.

[0278] In some embodiments, the endonuclease is an MG87 endonuclease (i.e., SEQ ID NO: 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%, 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 with any of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277.In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277. In some embodiments, the endonuclease comprises a sequence having 100% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277.

[0279] In some embodiments, the endonuclease is an MG88 endonuclease (i.e., SEQ ID NO: 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 with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with any of SEQ ID NO: 661-678 and 1278-1282. In some embodiments, the endonuclease comprises a sequence having 100% identity with any of SEQ ID NO: 661-678 and 1278-1282.

[0280] In some embodiments, the endonuclease is the 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 with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 659. In some embodiments, the endonuclease comprises a sequence having 100% identity with SEQ ID NO: 659.

[0281] In some embodiments, the endonuclease is an MG18 endonuclease (i.e., SEQ ID NO: 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 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 with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with any of SEQ ID NO: 696-698. In some embodiments, the endonuclease comprises a sequence having 100% identity with any of SEQ ID NO: 696-698.

[0282] In some embodiments, the endonuclease is an MG46 endonuclease (i.e., SEQ ID NO: 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 with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with any of SEQ ID NO: 975-981. In some embodiments, the endonuclease comprises a sequence having 100% identity with any of SEQ ID NO: 975-981.

[0283] In some embodiments, the endonuclease is the 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 with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 70% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266. In some embodiments, the endonuclease comprises a sequence having 100% identity with either SEQ ID NO: 1265 or SEQ ID NO: 1266.

[0284] In some embodiments, the endonuclease comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is located at the N-terminus of the endonuclease. In some embodiments, the NLS is located at the C-terminus of the endonuclease. In some embodiments, the NLS is located at both the N-terminus and C-terminus of the endonuclease.

[0285] In some embodiments, the NLS comprises a sequence of any of SEQ ID NO: 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 with any of SEQ ID NO: 1-46. In some cases, the NLS comprises a sequence having at least about 80% identity with SEQ ID NO: 1-46. In some cases, the NLS comprises a sequence having at least about 85% identity with SEQ ID NO: 1-46. In some cases, the NLS comprises a sequence having at least about 90% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 91% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 92% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 93% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 94% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 95% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 96% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 97% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 98% identity with SEQ ID NO: 1-46. In some cases, the NLS contains a sequence having at least about 99% identity with SEQ ID NO: 1-46. In some cases, NLS contains sequences that are 100% identical to SEQ ID NO: 1-46.

[0286] Table 1: Example NLS sequences that can be used with the Cas effector according to this disclosure

[0287]

[0288]

[0289] Guide polynucleotides

[0290] In some embodiments, the engineered nuclease system disclosed herein comprises engineered guide polynucleotides, such as guide ribonucleic acid (gRNA), single gRNA, or dual guide RNA.

[0291] In some embodiments, the guide RNA includes various structural elements, including, but not limited to: a spacer sequence that binds to the original spacer sequence (target sequence), crRNA, and optionally tracrRNA. In some embodiments, the guide RNA includes crRNA comprising a spacer sequence. In some embodiments, the guide RNA includes tracrRNA or modified tracrRNA.

[0292] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said 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%. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 70% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with any of SEQ ID NO: 710-722.In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 95% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 96% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 97% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 98% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 99% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having 100% identity with any of SEQ ID NO: 710-722.

[0293] In some cases, the tracrRNA contains 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 with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 70% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 75% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 80% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 85% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 90% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 91% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 92% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 93% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 94% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 95% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 96% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 97% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 98% identity with any of SEQ ID NO: 710-722. In some cases, the tracrRNA contains a sequence having at least about 99% identity with any of SEQ ID NO: 710-722.In some cases, tracrRNA contains a sequence that is 100% identical to any of SEQ ID NO: 710-722.

[0294] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said 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%. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 70% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with any of SEQ ID NO: 726-744.In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 95% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 96% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 97% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 98% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 99% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having 100% identity with any of SEQ ID NO: 726-744.

[0295] In some cases, the tracrRNA contains 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 with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 70% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 75% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 80% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 85% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 90% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 91% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 92% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 93% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 94% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 95% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 96% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 97% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 98% identity with any of SEQ ID NO: 726-744. In some cases, the tracrRNA contains a sequence having at least about 99% identity with any of SEQ ID NO: 726-744.In some cases, tracrRNA contains a sequence that is 100% identical to any of SEQ ID NO: 726-744.

[0296] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said 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%. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 70% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with any of SEQ ID NO: 745-767.In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 95% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 96% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 97% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 98% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 99% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having 100% identity with any of SEQ ID NO: 745-767.

[0297] In some cases, the tracrRNA contains 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 with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 70% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 75% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 80% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 85% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 90% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 91% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 92% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 93% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 94% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 95% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 96% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 97% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 98% identity with any of SEQ ID NO: 745-767. In some cases, the tracrRNA contains a sequence having at least about 99% identity with any of SEQ ID NO: 745-767.In some cases, tracrRNA contains a sequence that is 100% identical to any of SEQ ID NO: 745-767.

[0298] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the 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 with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 70% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 95% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 96% identity with SEQ ID NO: 699.In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 97% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 98% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having at least about 99% identity with SEQ ID NO: 699. In some cases, the tracrRNA comprises a sequence of at least about 60-100 consecutive nucleotides, the sequence having 100% identity with SEQ ID NO: 699.

[0299] In some cases, the tracrRNA contains 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 with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence having at least about 70% identity with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence having at least about 75% identity with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence having at least about 80% identity with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 85% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 90% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 91% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 92% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 93% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 94% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 95% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 96% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence with at least about 97% identity to SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence that has at least about 98% identity with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence that has at least about 99% identity with SEQ ID NO: 699. In some cases, the tracrRNA contains a sequence that has 100% identity with SEQ ID NO: 699.

[0300] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said 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%. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 70% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with any of SEQ ID NO: 700-702.In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 95% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 96% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 97% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 98% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 99% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having 100% identity with any of SEQ ID NO: 700-702.

[0301] In some cases, the tracrRNA contains 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 with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 70% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 75% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 80% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 85% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 90% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 91% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 92% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 93% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 94% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 95% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 96% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 97% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 98% identity with any of SEQ ID NO: 700-702. In some cases, the tracrRNA contains a sequence having at least about 99% identity with any of SEQ ID NO: 700-702.In some cases, tracrRNA contains a sequence that is 100% identical to any of SEQ ID NO: 700-702.

[0302] In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said 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%. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 70% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 75% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 80% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 85% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 90% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 91% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 92% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 93% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 94% identity with any of SEQ ID NO: 703-709.In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 95% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 96% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 97% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, the sequence having at least about 98% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having at least about 99% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA comprises a sequence consisting of at least about 60-100 consecutive nucleotides, said sequence having 100% identity with any of SEQ ID NO: 703-709.

[0303] In some cases, the tracrRNA contains 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 with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 70% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 75% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 80% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 85% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 90% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 91% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 92% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 93% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 94% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 95% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 96% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 97% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 98% identity with any of SEQ ID NO: 703-709. In some cases, the tracrRNA contains a sequence having at least about 99% identity with any of SEQ ID NO: 703-709.In some cases, tracrRNA contains a sequence that is 100% identical to any of SEQ ID NO: 703-709.

[0304] In some embodiments, the engineered nuclease system disclosed herein comprises engineered guide polynucleotides, such as guide ribonucleic acid (gRNA), single gRNA, or dual guide RNA.

[0305] 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 with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1137-1144.In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1137-1144. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any of SEQ ID NO: 1137-1144.

[0306] 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 with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1153-1156.In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1153-1156. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any of SEQ ID NO: 1153-1156.

[0307] 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 with any of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 92% identity with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 927-942, 945-950, 961-962, 969-970, 1123-1137, and 2433-2434.

[0308] 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 with any 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 with any 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 with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 843-880 and 1145-1152.In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 843-880 and 1145-1152. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any of SEQ ID NO: 843-880 and 1145-1152.

[0309] 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 with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 881-926.In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any of SEQ ID NO: 881-926. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with 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 with 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 with 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 with 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 with 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 with SEQ ID NO: 973 or SEQ ID NO: 974. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with SEQ ID NO: 973 or SEQ ID NO: 974.

[0310] 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 with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 75% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 91% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 92% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 93% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 94% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 957-960.In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 957-960. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any of SEQ ID NO: 957-960.

[0311] 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 with any 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 with any 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 943-944, 951-956, 965-968, and 971-972. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% identity with any one of SEQ ID NO: 943-944, 951-956, 965-968, and 971-972.

[0312] In some embodiments, the engineered guide polynucleotide comprises a synthetic nucleotide or a modified nucleotide. In some embodiments, the engineered guide polynucleotide comprises one or more nucleoside bonds modified from a natural phosphodiester. In some embodiments, all nucleoside bonds or their sequential nucleotide sequences of the engineered guide polynucleotide are modified. For example, in some embodiments, the nucleoside bonds contain sulfur (S), such as phosphate thioester nucleoside bonds.

[0313] In some embodiments, the engineered guide polynucleotide comprises a modification of the 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 compared to the ribose moiety present in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is within a ribocycle structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring (e.g., locked nucleic acid (LNA)) having a biradical bridge between the C2 and C4 carbons on the ribocycle, or an unconnected ribocycle (e.g., UNA) typically lacking a bond between the C2 and C3 carbons. In some embodiments, the sugar-modified nucleoside comprises a bicyclic hexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0314] In some embodiments, the engineered guide polynucleotide comprises one or more modified sugars. In some embodiments, sugar modification comprises modification by changing a substituent on the ribose ring to a group other than hydrogen or a 2'-OH group naturally present in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', or 5' position or a combination thereof. In some embodiments, the nucleoside having the modified sugar moiety comprises a 2'-modified nucleoside, for example, a 2'-substituted nucleoside. In some embodiments, the 2' sugar-modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (a 2'-substituted nucleoside) or comprising a 2'-linked diradical, and comprises a 2'-substituted nucleoside and an LNA (2'-4' diradical-bridged) nucleoside. Examples of 2'-substituted modified nucleosides include, 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, modification of the ribose group includes modification at the 2' position of the ribose group. In some embodiments, 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).

[0315] 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 some embodiments, the engineered guide polynucleotide comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a disaccharide. In some embodiments, the modified sugar comprises 2'-O-methoxyethyl. In some embodiments, the engineered guide polynucleotide comprises both nucleoside indirect head modification and nucleoside modification.

[0316] In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a polynucleotide sequence of a eukaryotic, fungal, plant, mammalian, or human genome. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a eukaryotic genome polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a fungal genome polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a plant genome polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a mammalian genome polynucleotide sequence. In some embodiments, the engineered guide polynucleotide comprises a sequence complementary to a human genome polynucleotide sequence.

[0317] 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 has a length of about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 1... 00, 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.

[0318] MG System

[0319] In some embodiments, this document describes engineered nuclease systems comprising a nuclease-containing and engineered guide polynucleotides. In some embodiments, the engineered guide polynucleotide comprises tracrRNA. In some embodiments, the engineered guide polynucleotide comprises a guide nucleic acid (e.g., gRNA). In polynucleotides, when referring to T, T means U (uracil) in RNA and T (thymine) in DNA.

[0320] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 710-722.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010, and 1263, and the engineered polynucleotide comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 710-722. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises one having 100% identity with any of SEQ ID NO: 49-51, 679-694, 983-1002, 1003-1010 and 1263, and the engineered polynucleotide comprises one having 100% identity with any of SEQ ID NO: 710-722.

[0321] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 726-744.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 659-660, 1158-1159, and 1267-1277, and the engineered polynucleotide comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 726-744. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises 100% identity with any of SEQ ID NO: 659-660, 1158-1159 and 1267-1277, and the engineered polynucleotide comprises 100% identity with any of SEQ ID NO: 726-744.

[0322] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 745-767.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 745-767. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises 100% identity with any of SEQ ID NO: 661-678 and 1278-1282, and the engineered polynucleotide comprises 100% identity with any of SEQ ID NO: 745-767.

[0323] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 70% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 75% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 80% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 85% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 90% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 95% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 96% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 97% identity with SEQ ID NO: 699.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 98% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises a sequence having at least about 99% identity with SEQ ID NO: 699. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises 100% identity with SEQ ID NO: 659, and the engineered polynucleotide comprises 100% identity with SEQ ID NO: 699.

[0324] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 75% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 700-702.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 700-702. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises 100% identity with any of SEQ ID NO: 696-698, and the engineered polynucleotide comprises 100% identity with any of SEQ ID NO: 700-702.

[0325] In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 70% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 70% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 75% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 75% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 80% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 80% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 85% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 85% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 90% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 90% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 95% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 95% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 96% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 96% identity with any of SEQ ID NO: 703-709.In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 97% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 97% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 98% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 98% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises a sequence having at least about 99% identity with any of SEQ ID NO: 975-981, and the engineered polynucleotide comprises a sequence having at least about 99% identity with any of SEQ ID NO: 703-709. In some embodiments, the engineered nuclease system comprises a nuclease and an engineered polynucleotide, wherein the nuclease comprises 100% identity with any of SEQ ID NO: 975-981; and the engineered polynucleotide comprises 100% identity with any of SEQ ID NO: 703-709.

[0326] cell

[0327] In some embodiments, this document describes a cell comprising the system described herein.

[0328] In some embodiments, the cells are eukaryotic cells (e.g., plant cells, animal cells, protist cells, or fungal cells), mammalian cells (Chinese hamster ovary (CHO) cells, young hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, mouse myeloma (NSO) cells, or human retinal cells), immortalized cells (e.g., HeLa cells, COS cells, HEK-293T cells, MDCK cells, 3T3 cells, PC12 cells, Huh7 cells, HepG2 cells, K562 cells, N2a cells, or SY5Y cells), insect cells (e.g., fall armyworm (Spodoptera frugiperda) cells, white armyworm (Trichoplusia ni) cells, Drosophila melanogaster (Drosophila melanogaster) cells, S2 cells, or Heliothis virescens (Heliothis virescens) cells), or yeast cells (e.g., Saccharomyces cerevisiae). The cells can be eukaryotic cells, cryptococcal cells, or Candida cells; plant cells (e.g., parenchyma cells, collenchyma cells, or sclerenchyma cells); fungal cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells, or Candida cells); or prokaryotic cells (e.g., Escherichia coli cells, Streptococcus cells, Streptomyces soil bacteria cells, or archaea cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0329] In some embodiments, the cells are 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 cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

[0330] Delivery and carrier

[0331] In some embodiments, this document discloses nucleic acid sequences encoding the engineered nuclease systems disclosed herein.

[0332] In some embodiments, the nucleic acid encoding the engineered nuclease system is DNA, such as linear DNA, plasmid DNA, or microcircular DNA. In some embodiments, the nucleic acid encoding the engineered nuclease system is RNA, such as mRNA.

[0333] 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., a circular DNA molecule that can autonomously replicate within a cell), a granule (e.g., a pWE or sCos vector), an artificial chromosome, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a phage particle, a phage derivative, a rod particle, or a virus. In some embodiments, the nucleic acid-based vector is selected from the list of the following: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-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.

[0334] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of mini-promoters, inducible promoters, constitutive promoters, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synaptic protein, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is the U6 promoter. In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter is encoded by any of SEQ ID NO: 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 with any of SEQ ID NO: 190-191.

[0335] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alpha virus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the virus is an alpha virus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is 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 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 a retrovirus.

[0336] 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-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, 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 derivatives thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0337] 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 some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 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 AAV15 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-rh10 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-rhM4-1 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. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0338] 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.

[0339] 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 lipids. In some embodiments, the lipid-associated nucleic acid may be encapsulated within the aqueous interior of a liposome, dispersed in a lipid bilayer of a liposome, linked to a liposome via a linker molecule associated with both the liposome and the nucleic acid, captured in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with lipids, incorporated with lipids, contained as a suspension in lipids, contained in or complexed with microparticles, or otherwise associated with lipids. In some embodiments, the nucleic acid is contained in lipid nanoparticles (LNPs).

[0340] In some embodiments, the fusion protein or genome editing system is introduced into cells stably or transiently in any suitable manner. In some embodiments, the fusion protein or genome editing system is transfected into cells. In some embodiments, cells are transduced or transfected with a nucleic acid construct encoding the fusion protein or genome editing system. For example, cells are transduced (e.g., with a virus encoding the fusion protein or genome editing system), or transfected with nucleic acid encoding the fusion protein or genome editing system or a translated fusion protein or genome editing system (e.g., with a plasmid encoding the fusion protein or genome editing system). In some embodiments, the transduction is stable or transient. In some embodiments, for example, when the fusion protein or genome editing system contains a CRISPR nuclease, cells expressing the fusion protein or genome editing system or containing the fusion protein or genome editing system are transduced or transfected with one or more gRNA molecules. In some embodiments, plasmids expressing the fusion protein or genome editing system are introduced into cells via electroporation, transient (e.g., lipid transfection), and stable genome integration (e.g., piggybac), and viral transduction (e.g., lentivirus or AAV), or other methods known to those skilled in the art. In some embodiments, the gene editing system is introduced into cells as one or more peptides. In some embodiments, delivery is achieved by using an RNP complex. Methods for delivering peptides and / or RNPs to cells are known in the art, such as by electroporation or by cell extrusion.

[0341] Exemplary methods for nucleic acid delivery include lipid transfection, nuclear transfection, electroporation, stable genome integration (e.g., piggybac), microinjection, gene guns (biolistics), virions, liposomes, immunoliposomes, polycationic or lipid-nucleic acid conjugates, naked DNA, artificial viral particles, and agent-enhanced DNA uptake. Lipid transfection is described, for example, in U.S. Patents 5,049,386; 4,946,787; and 4,897,355, and lipid transfection reagents are commercially available (e.g., Transfectam™, Lipofectin™, and SF cell line 4D-nuclear transfectant X Kit™ (Lonza Corporation)). Cationic and neutral lipids suitable for highly efficient receptor recognition of polynucleotides include cationic and neutral lipids as described in WO 91 / 17424 and WO 91 / 16024. In some embodiments, delivery is made 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 contained in liposomes or nanoparticles that specifically target host cells.

[0342] Other methods for delivering nucleic acids into cells are known to those skilled in the art. See, for example, US2003 / 0087817.

[0343] How to use

[0344] In some embodiments, methods for modifying target nucleic acids are described herein, the methods comprising providing an engineered nuclease system disclosed herein. In some embodiments, the engineered nuclease system comprises a nuclease 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.

[0345] In some embodiments, these methods are used to introduce modifications into the genome of a cell. In some embodiments, the modification is an insertion, deletion, or mutation. In some embodiments, these methods are used to introduce site-directed insertions, deletions, and / or mutations (e.g., insertions and mutations) into the genome of a cell. In some embodiments, these methods are used in combination with a nucleic acid template to facilitate site-directed insertion into the genome of a cell.

[0346] In some embodiments, the cell is a human cell. In some embodiments, the cell genome or a vector contained in the cell is modified. In some embodiments, the cell genome is modified in vitro. In some embodiments, the cell genome is modified in vivo.

[0347] In some embodiments, the engineered guide polynucleotide targets genes in cells. In some embodiments, the engineered guide polynucleotide targets genes in mammalian cells. In some embodiments, the mammalian cells are pig, cow, goat, sheep, rodent, rat, mouse, non-human primate, or human cells.

[0348] 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 with SEQ ID NO: 47. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 1283-1392 and 1502-1509.In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1283-1392 and 1502-1509. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1283-1392 and 1502-1509.

[0349] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 57-58, 101-119, 163-181, 225-243, 343-374, 421-433, 461-472, 499-511, 539-551, and 1098-1102.

[0350] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with the AAVS1 gene. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 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 with a sequence having at least about 70% identity with any one of SEQ ID NO: 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 with a sequence having at least about 75% identity with any one of SEQ ID NO: 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 with a sequence having at least about 80% identity with any one of SEQ ID NO: 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 with a sequence having at least about 85% identity with any one of SEQ ID NO: 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 with a sequence having at least about 90% identity with any of SEQ ID NO: 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 with a sequence having at least about 91% identity with any one of SEQ ID NO: 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 with a sequence having at least about 92% identity with any one of SEQ ID NO: 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 with a sequence having at least about 93% identity with any one of SEQ ID NO: 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 with a sequence having at least about 94% identity with any one of SEQ ID NO: 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 with a sequence having at least about 95% identity with any one of SEQ ID NO: 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 with a sequence having at least about 96% identity with any one of SEQ ID NO: 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 with a sequence having at least about 97% identity with any of SEQ ID NO: 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 with a sequence having at least about 98% identity with any one of SEQ ID NO: 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 with a sequence having at least about 99% identity with any one of SEQ ID NO: 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 with a sequence having 100% identity with any of SEQ ID NO: 59-60, 120-138, 182-200, 244-262, 375-406, 434-446, 473-484, 512-524, 552-564, and 1248-1252.

[0351] In some embodiments, the target gene is TRAC. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 92% identity with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 77-88, 139-150, 201-212, 407-413, 447-453, 485-491, 525-531, and 1096-1097.

[0352] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with 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. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 70% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 75% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 80% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 85% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 90% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 91% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 92% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 93% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 94% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 95% identity with any one of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 96% identity with any of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 97% identity with any of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 98% identity with any of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247. In some embodiments, the gRNA hybridizes with a TRAC sequence having at least about 99% identity with any of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538, and 1246-1247.In some embodiments, the gRNA hybridizes with a TRAC sequence that is 100% identical to any of SEQ ID NO: 89-100, 151-162, 213-224, 414-420, 454-460, 492-498, 532-538 and 1246-1247.

[0353] In some embodiments, the target gene is the B2M gene. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NOs: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 565-578 and 1095.In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 565-578 and 1095. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 565-578 and 1095.

[0354] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with 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. In some embodiments, the gRNA hybridizes with a B2M sequence having at least about 70% identity with any of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M sequence having at least about 75% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 80% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 85% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 90% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 91% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 92% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 93% identity with any of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 94% identity with any of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 95% identity with any of SEQ ID NOs: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M gene sequence having at least about 96% identity with any of SEQ ID NOs: 579-592 and 1245.In some embodiments, the gRNA hybridizes with a B2M sequence having at least about 97% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M sequence having at least about 98% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M sequence having at least about 99% identity with any of SEQ ID NO: 579-592 and 1245. In some embodiments, the gRNA hybridizes with a B2M sequence having 100% identity with any of SEQ ID NO: 579-592 and 1245.

[0355] In some embodiments, the target gene is the HBB gene. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 593-625.In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 593-625. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 593-625.

[0356] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with the HBB gene. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 626-658.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 626-658. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 626-658.

[0357] In some embodiments, the target gene is the albumin gene. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1083-1094.In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1083-1094. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1083-1094.

[0358] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with the albumin gene. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 1233-1244.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 1233-1244. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 1233-1244.

[0359] In some embodiments, the target gene is ATP7B. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1906-1931.In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1906-1931. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1906-1931.

[0360] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with ATP7B. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 1206-1231.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 1206-1231. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 1206-1231.

[0361] In some embodiments, the target gene is the PAH gene. In some embodiments, the gRNA is engineered to function in conjunction with an MG71 endonuclease (e.g., MG71-2; SEQ ID NO: 50), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1113-1122.In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1113-1122. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1113-1122.

[0362] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG71 nuclease) hybridizes with the PAH gene. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 1253-1262.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 1253-1262. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 1253-1262.

[0363] In some embodiments, the target gene is TRAC. In some embodiments, the gRNA is engineered to function in conjunction with an MG73 endonuclease (e.g., MG73-1; SEQ ID NO: 51), the endonuclease comprising 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 51 or SEQ ID NO: 1264. In some cases, 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 with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with SEQ ID NO: 61. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with SEQ ID NO: 61.In some embodiments, the gRNA comprises a sequence that is 100% identical to SEQ ID NO: 61.

[0364] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG73 nuclease) hybridizes with TRAC. In some cases, the gRNA hybridizes with 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 with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 75% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with SEQ ID NO: 62. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with SEQ ID NO: 62.

[0365] In some embodiments, the target gene is TRAC. In some embodiments, the gRNA is engineered to function in conjunction with the MG89 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with 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 with any of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NOs: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any one of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 63-69 and 263-302.In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 63-69 and 263-302. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 63-69 and 263-302.

[0366] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG89 nuclease) hybridizes with TRAC. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 75% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 70-76 and 303-342.In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 70-76 and 303-342. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 70-76 and 303-342.

[0367] In some embodiments, the target gene is TRAC. In some embodiments, the gRNA is engineered to function in conjunction with the MG87 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NOs: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1011-1019 and 1050.In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1011-1019 and 1050. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1011-1019 and 1050.

[0368] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG87 nuclease) hybridizes with TRAC. In some embodiments, the gRNA hybridizes with 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 with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 75% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NOs: 1161-1169 and 1200.In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NOs: 1161-1169 and 1200. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NOs: 1161-1169 and 1200.

[0369] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, the gRNA is engineered to function in conjunction with the MG87 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with any of SEQ ID NO: 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 with any of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NOs: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1020-1049 and 1051-1055.In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1020-1049 and 1051-1055. In some embodiments, the gRNA comprises a sequence that is 100% identical to any one of SEQ ID NO: 1020-1049 and 1051-1055.

[0370] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG87 nuclease) hybridizes with the AAVS1 gene. In some embodiments, the gRNA hybridizes with 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 with any of the SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the SEQ ID NOs: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 1170-1205.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 1170-1205. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 1170-1205.

[0371] In some embodiments, the target gene is the ATXN2 gene. In some embodiments, the gRNA is engineered to function in conjunction with the MG21 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with 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 with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1786-2045.In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1786-2045. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1786-2045.

[0372] In some embodiments, the gRNA (e.g., functioning in conjunction with the MG87 nuclease) hybridizes with the ATXN2 gene. In some embodiments, the gRNA hybridizes with 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 with any of the sequences in SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 70% identity with any of the sequences in SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 80% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 85% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 90% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 91% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 92% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 93% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 94% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 95% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 96% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 97% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having at least about 98% identity with any of SEQ ID NO: 1526-1785.In some embodiments, the gRNA hybridizes with a sequence having at least about 99% identity with any of SEQ ID NO: 1526-1785. In some embodiments, the gRNA hybridizes with a sequence having 100% identity with any of SEQ ID NO: 1526-1785.

[0373] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, the gRNA is engineered to function in conjunction with the MG23 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with 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 with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 75% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any of SEQ ID NO: 1393-1493.In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any of SEQ ID NO: 1393-1493. In some embodiments, the gRNA comprises a sequence having 100% identity with any of SEQ ID NO: 1393-1493.

[0374] In some embodiments, the target gene is the AAVS1 gene. In some embodiments, the gRNA is engineered to function in conjunction with the MG23 endonuclease, which 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 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with 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 with any of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 70% identity with any of SEQ ID NOs: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 80% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 85% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 90% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 91% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 92% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 93% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 94% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525.In some embodiments, the gRNA comprises a sequence having at least about 95% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 96% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 97% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 98% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence having at least about 99% identity with any one of SEQ ID NO: 1494-1501 and 1510-1525. In some embodiments, the gRNA comprises a sequence that is 100% identical to any one of SEQ ID NO: 1494-1501 and 1510-1525.

[0375] Example

[0376] Example 1 - Gene editing results of TRAC and AAVS1 at the DNA level in K562 cells

[0377] MG71-2 mRNA, along with matching guide RNA (500 ng mRNA / 150 pmol guide), was nuclearly transfected into K562 cells (200,000). Cells were harvested, and genomic DNA was prepared three days post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. Amplicones were sequenced and analyzed to measure gene editing (…). Figure 1 ).

[0378] Example 2 - Gene editing results of TRAC at the DNA level in K562 cells

[0379] MG89-2 mRNA, along with matching guide RNA (500 ng mRNA / 150 pmol guide), was nuclearly transfected into K562 cells (200,000). Cells were harvested, and genomic DNA was prepared three days post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. Amplicones were sequenced and analyzed to measure gene editing (…). Figure 2 ).

[0380] Example 3 - Analysis of gene editing results of MG71-2 scaffold using guide hAAVS1 C3 at the DNA level

[0381] Use 500 ng mRNA / 150 pmol guide, 250 ng mRNA / 75 pmol guide, or 125 ng mRNA / 37.5 pmol guide for 1.2 x 10⁻⁶ mRNA. 5 K562 cells were nuclear transfected. Cells were collected, and genomic DNA was prepared three days post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences of each guide RNA. Amplicones were sequenced and analyzed to measure gene editing (…). Figure 3A For the second round of engineering of the MG71-2 scaffold at the DNA level using the guide hAAVS1 C3, 500 ng mRNA / 150 pmol guide was used to target 1.2 x 10⁻⁶ scaffolds. 5 K562 cells were nuclear transfected and then processed in the downstream steps described above. Figure 3B ).

[0382] Example 4 - Analysis of gene editing results at the DNA level using the original versus optimized MG71-2 scaffold

[0383] Use 500 ng mRNA / 150 pmol guide on 1.2 x 10 5 K562 cells were nuclear transfected and processed for downstream steps. Spacers indicated on the Y-axis were tested using 22 nt or 24 nt spacers and original or shortened MG71-2 scaffolds. Figure 4 ).

[0384] Example 5 - Analysis of gene editing results at the DNA level using the original versus optimized MG71-2 scaffold

[0385] Use 500 ng mRNA / 150 pmol guide on 1.2 x 10 5 K562 cells were nuclear transfected. Cells were collected, and genomic DNA was prepared three days post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences of each guide RNA. Amplicones were sequenced and analyzed to measure gene editing. Figure 5A The results of editing at exon 2 of the human B2M gene using the MG71-2 engineering wizard (scaffold 21) are shown. Figure 5B The results of editing at exon 3 of the human HBB gene using the MG71-2 engineering wizard (scaffold 21) are shown.

[0386] Example 6 - Computer simulation identification of type II CRISPR effectors

[0387] Computer simulation identification of type II CRISPR effectors

[0388] The proposed type II CRISPR effectors were identified by searching a wide range of databases. Homologous compounds were filtered to include those with an e-value ≤ 1. -5 Homologous genes with a length ≥500 aa and containing a relevant CRISPR locus predicted using minCED were selected. Effectors were deduplicated at 99% amino acid identity (AAI), globally aligned, and a phylogenetic tree was constructed.

[0389] Ancestor Reconstruction of MG71 Nuclease

[0390] To generate further diversity within the MG71 family of type II nucleases, the Ancestor Sequence Reconstruction (ASR) algorithm was used. ASR is a computational technique for reconstructing potential ancestral sequences from ancient organisms using existing protein sequences and inferred relationships between them. 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 constructed. Two type II-B sequences were used as outgroups to root the tree. Sequence reconstruction was performed. For each reconstructed node, insertions and deletions were manually identified. Four ancestral sequences were reconstructed with high confidence: MG71-40, MG71-41, MG71-42, and MG71-43. Figure 6 (SEQ ID NO: 691-694).

[0391] result

[0392] Type II effectors were identified from the MG71, MG87, and MG88 families (SEQ ID NO: 659-690). All type II effectors possess the catalytic residues required for activity and have lengths ranging from 1,031 aa to 1,438 aa.

[0393] Example 7 - sgRNA design and in vitro activity of MG71, MG73, MG74, MG87 and MG88 nucleases

[0394] sgRNA design

[0395] Predicting tracrRNAs. TracrRNAs (SEQ ID NO: 699-767) and repetitive sequences (SEQ ID NO: 768-836) were folded, trimmed, and linked to a four-loop sequence GAAA or TTCG (if GAAA was altered). Effectors were screened using multiple sgRNA designs (SEQ ID NO: 837-974) consisting of short and long scaffolds with multiple spacers of varying lengths (20, 22, and 24 nt) and GC contents (40% and 67%).

[0396] In vitro activity assay

[0397] Nucleases were expressed using PCR-generated templates at 5–10 nM. Following expression, the nucleases were diluted 10-fold and incubated for 1 hour in a mixture of 5 nM 8N protospacer adjacent motif (PAM) library plasmid and 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCl2 containing 50 nM sgRNA. The plasmid digest was cleaned with SPRI beads and eluted in TE buffer. The digested PAM plasmid (15 nM) was ligated to blunt ends (NEB) of a double-stranded adaptor oligonucleotide (150 nM) using T4 ligase in 1X T4 ligase buffer. The ligated products were sequenced using 150 bp single-read amplicon sequencing. Reads were filtered by a quality score >20. PAMs were identified by mapping the reads to the required PAM plasmid backbone for a perfect match. A SeqLogo for the PAM was generated, and the PAM sequence was determined by the height of each nucleotide. The cleavage site is identified by calculating the distance between the PAM and the connected joiner.

[0398] result

[0399] Multiple candidates from each family were active, demonstrating site-directed nuclease activity with their respective sgRNA designs. The cleavage sites and PAMs identified based on NGS data are summarized in Tables 2-6, and example PAM SeqLogos are shown in [Table 2-6]. Figure 7-11 Each family possesses a variety of PAMs, demonstrating the versatility of these systems as potential gene-editing agents. Nucleases derived from metagenomic data from the MG71 family, including the MG18 and MG46 families, exhibit a range of PAM specificities, with a preference for A at position 4 (…). Figure 7 The MG74 family, including the MG17, has PAM rich in A / C ( ). Figure 9 The MG87 nuclease family exhibits the strongest preference for the 5th and 6th bases from the spacer. Figure 10 The MG88 family tends to have purine-rich PAMs at the second to fourth positions. Figure 11 ).

[0400] Table 2: Summary of the activity of the MG71 family

[0401]

[0402]

[0403] Table 3: Summary of the activity of the MG73 family

[0404]

[0405] Table 4: Summary of the activity of the MG74 family

[0406]

[0407] Table 5: Summary of the activity of the MG87 family

[0408]

[0409] Table 6: Summary of the activity of the MG88 family

[0410]

[0411] Additional candidates were constructed using ancestral sequence reconstruction. Ancestors MG71-42 and MG71-43 shared 91.0% and 81.1% AAI with MG71-2, respectively. Two of the three test candidates showed robust activity against MG71-2, MG71-1, or MG18-1 sgRNA. Figure 12A-12B (See Table 7). The resulting PAM from MG71-42 is similar to that of the natural metagenomic nuclease MG71-2. The PAM from MG71-43 is more relaxed in vitro than that from MG71-2, but the NNNACT of the MG71-2 PAM is a subset of the observed PAM.

[0412] Table 7: Summary of ASR activity of MG71

[0413]

[0414] Example 8 - Activity of MG71-2 gRNA in mammalian cells

[0415] To further test the activity of MG71-2, a guide RNA targeting the region of the human AAVS1 locus was designed using in vitro PAM with MG71-2. 50,000 K562 cells were nuclear transfected with 500 ng MG71-2 mRNA and 150 pmol of chemically synthesized guide RNA in 96-well plates using the manufacturer's recommended cell type-specific procedure. gDNA was extracted 72 hours post-transfection. PCR primers suitable for NGS-based DNA sequencing were generated, optimized, and used to amplify the target sequence. The amplicon was sequenced and analyzed to measure gene editing.

[0416] result

[0417] Several guides showed >50% activity, further supporting the robust activity of the nuclease. Figure 13 ).

[0418] Example 9 - In vitro activity of a type II CRISPR system with appropriate point mutations

[0419] Ancestor sequence reconstruction (ASR) to generate MG71 variant

[0420] To generate further diversity within the MG71 family of type II nucleases, the Ancestor Sequence Reconstruction (ASR) algorithm, as described in Example 6 above, was used. For this analysis, MAFFT was used to align 432 type II nuclease sequences with parameters, constructing a phylogenetic tree and reconstructing ancestral nodes. Eight ancestral nodes were reconstructed: MG71-88, MG71-89, MG71-90, MG71-91, MG71-92, MG71-95, MG71-96, MG71-97 (…). Figure 14 (SEQ ID NO: 1003-1010).

[0421] In vitro activity assay

[0422] As described in Example 7 above, a nuclease was expressed using a template generated by 5–15 nM PCR with a T7 promoter. After expression, the nuclease was diluted 10-fold and digested for 1 hour in a mixture containing a 5 nM spacer adjacent motif (PAM) library plasmid and 50 nM sgRNA in 10 mM Tris pH 7.5, 100 mM NaCl, and 10 mM MgCl2. The digest was cleaned with SPRI beads and eluted in TE buffer. The digested PAM plasmid (15 nM) was ligated to blunt ends (NEB) of a double-stranded adaptor oligonucleotide (150 nM) using T4 ligase in 1X T4 ligase buffer. The ligated product was amplified using an NGS adaptor and sequenced using 300 bp single-read amplicon sequencing. Reads were filtered by a quality score > 20. PAMs were identified by mapping the reads to the required PAM plasmid backbone for a perfect match. The SeqLogo of the PAM is generated, and the PAM sequence is determined by the height of each nucleotide. The cleavage site is identified by calculating the distance between the PAM and the linked adaptor.

[0423] result

[0424] The predicted structure of Alphafold2 based on MG71-2 overlaps with the structure of SpCas9 (PDB ID: 4UN3), and 16 residues within the first interaction shell of the PAM base pair are selected. Figure 15 To alter the PAM sequence of MG71-2, 16 residues were mutated to allow the insertion or removal of positively charged residues. Similar residues were identified in MG71-43 (SEQ ID NO: 694) to generate four MG...

Claims

1. An engineered nuclease system, comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 70% sequence identity with any one of SEQ ID NO: 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 hybridize with a target nucleic acid sequence.

2. The engineered nuclease system according to claim 1, wherein the endonuclease comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO:47-52, 659-698, 983-1010, 1263-1282, 1158-1159 and 975-981.

3. The engineered nuclease system according to claim 1, wherein the endonuclease comprises a sequence having 90% sequence identity with any one of SEQ ID NO:47-52, 659-698, 983-1010, 1263-1282, 1158-1159 and 975-981.

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

5. The engineered nuclease system according to claim 4, wherein the tracrRNA comprises a sequence having at least 90% sequence identity with any one of SEQ ID NO: 710-722, 726-744, 745-767, 699, 700-702 and 703-709.

6. The engineered nuclease system according to claim 4, wherein the tracrRNA comprises a sequence having 100% sequence identity with any one of SEQ ID NO: 710-722, 726-744, 745-767, 699, 700-702 and 703-709.

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

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

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

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

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

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

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

14. An engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 47; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1137-1144, 1283-1392, 1502-1509 and 1786-2045.

15. An engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 48; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 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 with any one of 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 with a target nucleic acid sequence, wherein the engineered guide polynucleotide comprises the sequences SEQ ID NO: 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, 565-57.

8. A sequence that has at least 80% sequence identity among any one of 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 with 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 with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with SEQ ID NO:

61.

18. An engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 659, 660, 1158, 1159 and 1267-1277; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 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 with any one of SEQ ID NO: 661-678 and 1278-1282; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 745-767 and 881-926.

20. An engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 659; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 699 and 973-974.

21. An engineered nuclease system, comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 696-698; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with either SEQ ID NO: 700-702 or 957-960.

22. An engineered nuclease system, comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 975-981; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 703-709, 943-944, 951-956, 965-968 and 971-972.

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

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

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

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

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

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

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

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

31. A method for modifying a target nucleic acid sequence, the method comprising contacting the target nucleic acid sequence with an engineered nuclease system according to any one of claims 1 to 30.

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

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

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

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

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

37. The method according to any one of claims 31 to 36, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1161-1262, 1526-1785 and 2046-2050.

38. A method for modifying a target nucleic acid sequence in mammalian cells, the method comprising contacting the mammalian cells with an engineered nuclease system according to any one of claims 1 to 30.

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

40. A method for modifying the hydroxy acid oxidase 1 (HAO1) ​​gene, the method comprising contacting the HAO1 gene with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 47; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 1283-1392 and 1502-1509.

41. The method of claim 40, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1283-1392 and 1502-1509.

42. A method for modifying ATPase copper transporter β (ATP7B), the method comprising contacting ATP7B with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of 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 with a target nucleic acid sequence.

43. The method of claim 42, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO:1056-1081.

44. The method of claim 42, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1206-1231.

45. A method for modifying adeno-associated virus integration site 1 (AAVS1), the method comprising contacting AAVS1 with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 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 with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 710-722.

46. ​​The method of claim 45, wherein the engineered guide polynucleotide comprises a sequence of any one of SEQ ID NO: 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 any one of SEQ ID NO: 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 for modifying the T cell receptor α constant (TRAC), the method comprising contacting the TRAC using an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease, said endonuclease comprising a sequence having at least 80% sequence identity with any one of SEQ ID NO: 49-51, 679-694, 983-1010, 1263, 48, 659, 660, 1158, 1159 and 1267-1277; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 710-722.

49. The method of claim 48, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 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 any one of SEQ ID NO: 62, 89-100, 151-162, 213-224, 414-420, 454-460, 492-489, 532-538, 1161-1168, and 1200.

51. A method for modifying an albumin gene, the method comprising contacting the albumin gene with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with 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 with a target nucleic acid sequence.

52. The method of claim 51, wherein the engineered guide polynucleotide comprises a sequence having any of 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 for modifying a β-2-microglobulin (B2M) gene, the method comprising contacting the B2M gene with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of 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 with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 710-722.

55. The method of claim 54, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 565-578 and 1095.

56. The method of claim 54, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 579-592 and 1245.

57. A method for modifying the hemoglobin subunit β (HBB) gene, the method comprising contacting the HBB gene with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of 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 with a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least 80% sequence identity with any of SEQ ID NO: 710-722.

58. The method of claim 57, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 593-625.

59. The method of claim 57, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 626-658.

60. A method for modifying a phenylalanine hydroxylase (PAH) gene, the method comprising contacting the PAH gene with an engineered nuclease system, the engineered nuclease system comprising: a) an endonuclease comprising a sequence having at least 80% sequence identity with any one of 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 with a target nucleic acid sequence.

61. The method of claim 60, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1113-1122.

62. The method of claim 60, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1253-1262.

63. A method for modifying the ataxia protein 2 (ATXN2) gene, the method comprising contacting the ATXN2 gene with an engineered nuclease system, the engineered nuclease system comprising: c) An endonuclease comprising a sequence having at least 80% sequence identity with SEQ ID NO: 47; and d) Engineered guide polynucleotides, wherein the engineered guide polynucleotides are configured to form a complex with the endonuclease and hybridize with the target nucleic acid sequence.

64. The method of claim 63, wherein the engineered guide polynucleotide comprises the sequence of any one of SEQ ID NO: 1786-2045 and 2051-2055.

65. The method of claim 63, wherein the target nucleic acid sequence comprises the sequence of any one of SEQ ID NO: 1526-1785 and 2046-2050.

66. A cell comprising an engineered nuclease system according to any one of claims 1 to 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 according to 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 according to claim 66, wherein the cell is 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 cells, HT1080, HepG2, Huh7, K562, primary cells or derivatives 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.

Images