Class 2, type v crispr systems

Abstract

Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.

Description

Attorney Docket No : 00010.036.1801CLASS 2, TYPE V CRISPR SYSTEMSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 753,419, filed on February 3, 2025; and to U.S. Provisional Patent Application No.63 / 941,152, filed on December 15, 2025, the disclosures of each of which are incorporated by reference herein in their entireties for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 29, 2026, is named 00010_036_1801_SL.xml and is 2,381,899 bytes in size.SUMMARY

[0003] Described herein, in certain embodiments, are engineered nuclease systems, comprising: a) an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202. M204, E236, E255, and N329 as compared to SEQ ID NO: 28; and b) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence. In some embodiments, the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises at least 90% sequence identity to SEQ ID NO: 1382. In some embodiments, the endonuclease comprises at least 95% sequence identity7to SEQ ID NO: 1382. In some embodiments, the endonuclease comprises SEQ ID NO: 1382.

[0004] Described herein, in certain embodiments, are engineered nuclease systems, comprising: a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease has at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381. In some embodiments, the endonuclease has at least 90% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381. In some embodiments, the endonuclease has 1 0% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381. In some embodiments, the engineered guide polynucleotide comprises a crRNA and a tracrRNA. In some embodiments, the engineered guide polynucleotide having at least 90% sequence identity to any one of SEQ ID NOs: 201, 424-449,- 1 - #592931Attorney Docket No : 00010.036.1801790-838, 888-903, 920-959, 1063-1078, 1079-1108, 1136-1165, 1209-1211, 1212-1293, 1413-1420, 122-155, 194-200, 202-215, 385-423, 450-504, 1047-1062, and 1387. In some embodiments, the engineered guide polynucleotide having 100% sequence identity to any one of SEQ ID NOs: 201, 424-449, 790-838, 888-903, 920-959, 1063-1078, 1079-1108, 1136-1165. 1209-1211, 1212-1293, 1413-1420, 122-155, 194-200, 202-215, 385-423, 450-504, 1047-1062, and 1387. In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the endonuclease binds non-covalently to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide. In some embodiments, the engineered nuclease systems further comprise a DNA methyltransferase. In some embodiments, the DNA methyltransferase binds non-covalently to the endonuclease. In some embodiments, the DNA methyltransferase is fused to the endonuclease in a single polypeptide. In some embodiments, the DNA methyltransferase comprises Dmnt3A or Dnmt3L.

[0005] Described herein, in certain embodiments, are methods of modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease systems described herein. In some embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the modification is in vitro. In some embodiments, the modification is in vivo. In some embodiments, the modification is ex vivo.

[0006] Described herein, in certain embodiments, are methods of modify ing a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease systems described herein. In some embodiments, the methods further comprise selecting cells comprising the modification.

[0007] Described herein, in certain embodiments, are methods of modifying AAVS1 comprising contacting AAVS1 using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 1413, 1212-1245, 920-959, 1082-1108, and 1136-1165.

[0008] Described herein, in certain embodiments, are methods of modify ing SOD1 comprising contacting SOD1 using an engineered nuclease system comprising: a) an endonuclease having at - 2 - #592931Attorney Docket No : 00010.036.1801least 70% sequence identity to any one of SEQ IDNOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 1246-1293.

[0009] Described herein, in certain embodiments, are methods of modifying a VCP gene comprising contacting the VCP gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify to SEQ ID NO: 28; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1387.

[0010] Described herein, in certain embodiments, are methods of modifying a VCP gene comprising contacting the VCP gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify to any one of SEQ ID NO: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1387.

[0011] Described herein, in certain embodiments, are methods of modifying a VEGFA gene comprising contacting the VEGFA gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify' to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1414.

[0012] Described herein, in certain embodiments, are methods of modifying a IFNG gene comprising contacting the IFNG gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify' to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1415.

[0013] Described herein, in certain embodiments, are methods of modifying a CLIC4 gene comprising contacting the CLIC4 gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1416.- 3 - #592931Attorney Docket No : 00010.036.1801

[0014] Described herein, in certain embodiments, are methods of modifying a FGF18 gene comprising contacting the FGF18 gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1417.

[0015] Described herein, in certain embodiments, are methods of modify ing a NLRC4 gene comprising contacting the NLRC4 gene using an engineered nuclease system comprising a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1418.

[0016] Described herein, in certain embodiments, are methods of modifying a NUDT16 gene comprising contacting the NUDT16 gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identity' to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1419.

[0017] Described herein, in certain embodiments, are methods of modifying a EMC6 gene comprising contacting the EMC6 gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identify to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1420.

[0018] Described herein, in certain embodiments, are nucleic acids encoding the engineered nuclease systems described herein.

[0019] Described herein, in certain embodiments, are cells comprising the engineered nuclease systems described herein. 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. In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof. In - 4 - #592931Attorney Docket No : 00010.036.1801some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is a T cell. In some embodiments, the primary cell is a hematopoietic stem cell (HSC).BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure’’ and “FIG.” herein), of which:

[0021] FIG. 1 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis). The scaffold length is represented by diagonal hatched (134 nt) and open (119 nt) bars (n = 2 or 3). The spacer length (nt) is shown on the X axis for each guide.

[0022] FIG.2 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis; bars). The percent out of frame (% OOF) total obtained as % OOF InDeis x % InDeis is shown as circles within the bars. Historical levels of editing efficiency are shown as a dotted line and were obtained with the MG119-28 nuclease and a sgRNA comprising the 119 nt scaffold and a 20 nt spacer targeting hAAVSl (SEQ ID NO: 1244). 52 single point mutants (SPMs) tested are shown on the X axis (protein variants).

[0023] FIG.3 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis; bars). The percent out of frame (% OOF) total obtained as % OOF InDeis x % InDeis is shown as circles within the bars. Historical levels of editing efficiency are shown as a dotted line and w ere obtained with the MG119-28 nuclease and a sgRNA comprising the 119 nt scaffold and a 20 nt spacer targeting hSODl (SEQ ID NO: 1248). 52 SPMs tested are shown on the X axis (protein variants).

[0024] FIGs. 4A-4B depict editing efficiency as percent NGS reads with insertions or deletions (InDeis). The MG119-28 WT and top performing SPMs are shown on the X axis (protein variants). The sgRNA tested comprised the 119 nt scaffold and a 20 nt spacer targeting the hSODl_D9 (SEQ ID NO: 1264; FIG. 4A) and hSODl_D10 (SEQ ID NO: 1256; FIG. 4B).

[0025] FIG.5 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis; bars). The percent out of frame (% OOF) total obtained as % OOF InDeis x % InDeis is shown as circles within the bars. WT levels of editing efficiency are shown as a dotted line and a bar. The experiment tested the sgRNAs with the 134 nt scaffold and 19 nt spacer (AAVS1_F4; SEQ ID NO: 1245). 34 double and triple mutants and a hexamutant were screened and are shown on the X axis as protein variants.- 5 - #592931Attorney Docket No : 00010.036.1801

[0026] FIG.6 depicts editing efficiency as percent NGS reads with insertions or deletions (InDeis; bars). The percent out of frame (% OOF) total obtained as % OOF InDeis x % InDeis is shown as circles within the bars. WT levels of editing efficiency are show n as a dotted line and a bar. This experiment tested the sgRNAs with the 134 nt scaffold and 19 nt spacer (SOD1_A9; SEQ ID NO: 1271). 34 double and triple mutants and a hexamutant were screened and are shown on the X axis as protein variants.

[0027] FIG. 7 shows the wild-ty pe (WT) and R155H mutation-bearing sequences for human IBMPFD patients (top) and the murine disease model cells used in these experiments (bottom), with corresponding amino acids (SEQ ID NOs: 1391-1394).

[0028] FIGs. 8A and 8B depict bar graphs showing % in del (FIG. 8A) and % of out-of-frame (OOF) indels (FIG.8B) for MG119-28 and guide gr2154 targeting the mutant VCP allele in mouse VCPR155H / +fibroblasts. Cas9 and guide gr2155 were used as a positive control.

[0029] FIG. 9 depicts alignment of sequencing reads produced by MG119-28 and guide gr2154 in fibroblasts derived from VCPR155H +mice. The mutated allele is shown as reference. The unedited, wild-type sequence is represented in 46.87% of sequencing reads, w hile the intact unedited R155H mutant sequence is present in 0.50% of sequencing reads.

[0030] FIG. 10 depicts a schematic illustration of an AAV construct design comprising MG119-28, nuclear EGFP fluorophore, and mouse guide RNA.

[0031] FIG. 11 depicts a schematic diagram outlining stereotactic delivery of MG119-28 and analysis of Atxn2 knockdown via confocal imaging or quantification of indels via NGS.

[0032] FIG. 12 depicts images of representative sections stained using methods described here and imaged on a confocal microscope. Staining corresponding to Hoechst or nuclear staining in the upper left image is indicated in dark grey / 1201, NLS::EGFP signal in the lower left panel is indicated in dark grey / 1202, V5-tagged MG119-28 signal in the lower right image is indicated in dark grey / 1203, and ATXN2 immunofluorescence signal in the upper right image is indicated in dark grey / 1204. Experimental and targeting sides are labeled in the image. The injection sites are labeled on the upper left image, with PIC54 / x«2 targeting guide injected in the left Ml region and control R26g4 guide injected into the right Ml region. 1201, 1202, 1203, and 1204 with arrows in white or black point to examples of staining.

[0033] FIG. 13 depicts a bar graph showing percentage insertion and deletion editing (% indel) in pre-FACS, GFP enriched, and GFP depleted samples.

[0034] FIG. 14 depicts a bar graph showing percentage out of frame (OOF) total editing in pre-FACS, GFP enriched, and GFP depleted samples.- 6 - #592931Attorney Docket No : 00010.036.1801

[0035] FIGs. 15A and 15B depict bar graphs showing normalized MG119-28 expression. The graphs correspond to either normalized MG119-28 expression (FIG. 15A) or normalized ATXN2 protein expression (FIG. 15B) in vivo as assessed by confocal imaging and immunohistochemistry after stereotactic delivery into the motor cortex region Ml . Both graphs display the data from single cells averaged per tissue section and then averaged again per animal. Each animal had 2-3 tissue sections averaged per animal with an average of 1000 cells quantified per section. Percentage ATXN2 knockdow n relative to the R26g4 control is displayed on the graph of FIG. 15B.

[0036] FIG. 16 depicts a plot of ATXN2 expression for ATXN2-targeting guide P1C5 versus control guide R26g4 across all V5+ cells. All cells quantified are displayed in the violin plots with average ATXN2 values displayed above each plot for each animal. Statistical analyses and comparisons were performed on data averaged per animal as described in FIGs. 11, 15A and 15B.

[0037] FIGs. 17A-17B depict graphs comparing the indel editing efficiencies of MG119-28 (A13Casl2f, upward diagonal hatched bar), OsCasl2f (downward diagonal hatched bar), and LbCasl2a (open bar) across eight TTTA-PAM containing genomic loci in K562 cells. Editing activity was assessed via NGS following transfection with mRNA encoding the respective nuclease and a 134 nt sgRNA scaffold paired with a 20 nt spacer. All tested nucleases recognize overlapping PAM sequences. Data are presented as the mean indel percentage (n = 3 independent biological replicates). FIG. 17B is a box-and-whisker plot comparing the indel editing efficiencies of the nucleases across the same eight different loci from FIG. 17A. The results indicate that MG119-28 can target six loci with higher activity than OsCasl2f and exhibits editing activity7comparable to LbCasl2a.

[0038] FIG. 18A is a plot comparing the indel editing efficiencies of MG119-28 (A13Casl2f, downward tight diagonal hatch) and A13Casl2f combinatorial mutant K93R-M204K-E236K (downward diagonal hatch) across eight TTTA-PAM containing genomic loci in K562 cells. Editing activity w as assessed via NGS following transfection with mRNA encoding the respective nuclease and a 134 nt sgRNA scaffold paired with a 20 nt spacer. Data are presented as the mean indel percentage (n = 3 independent biological replicates). FIG. 18B is a box-and-whisker plot comparing indel editing efficiencies of the wild type and RKK variant. The engineered variant showed a mean 2.4-fold enhancement across all eight loci, with a maximum improvement of 26-fold observed at EMC6.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0039] The Sequence Listing filed herewith provides exemplary polynucleotide and polypeptide sequences for use in methods, compositions, and systems according to the disclosure. Below are exemplary descriptions of sequences therein.- 7 - #592931Attorney Docket No : 00010.036.1801

[0040] SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 show the full-length amino acid sequences of MG119 nucleases.

[0041] SEQ ID NO: 28 shows the amino acid sequence of MG119-28 nuclease. SEQ ID NO: 1382 shows the amino acid sequence of K93R-M204K-E236K (“RKK") variant of MG119-28 nuclease.

[0042] SEQ ID NO: 1398 shows the nucleotide sequence of an engineered open reading frame of V5-tagged MG119-28.

[0043] SEQ ID NOs: 122-155 show nucleotide sequences of MG119 tracrRNAs derived from the same loci as a MG119 Cas effector.

[0044] SEQ ID NOs: 156-175 show nucleotide sequences of MG119 minimal arrays.

[0045] SEQ ID NOs: 176-181 show nucleotide sequences of MG119 target CRISPR repeats.

[0046] SEQ ID NO: 1204 is a direct repeat of MG119-1.MG 119 guide RNAs

[0047] SEQ ID NOs: 194-215. 385-504, 1047-1081, and 1209-1211 show nucleotide sequences of MG119 sgRNAs.MG 119-1 guide RNA

[0048] SEQ ID NO: 1205 is a single guide RNA designed for MG119-1.MG119-28 guide RNAs

[0049] SEQ ID NOs: 365-368, 790-838, 1166. 1168, 1170, and 1172 show the nucleotide sequences of sgRNAs engineered to function with an MG119-28 nuclease in order to target an albumin gene.APOA 1 Targeting with MG 119-28

[0050] SEQ ID NOs: 888-903 show the nucleotide sequences of sgRNAs engineered to function with an MG119-28 nuclease in order to target APOA1.AAVS1 Targeting with MG 119-28

[0051] SEQ ID NOs: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413 show the nucleotide sequences of sgRNAs engineered to function with an MG119-28 nuclease in order to target AAVS1.SOD! Targeting with MG119-28

[0052] SEQ ID NOs: 1246-1293 show the nucleotide sequences of MG119-28 hSODl guide RNA. VEGFA Targeting with MG 119-28

[0053] SEQ ID NO: 1414 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target VEGFA.- 8 - #592931Attorney Docket No : 00010.036.1801IFNG Targeting with MG119-28

[0054] SEQ ID NO: 1415 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target IFNG.CLIC4 Targeting with MG 119-28

[0055] SEQ ID NO: 1416 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target CLIC4.FGF18 Targeting with MG119-28

[0056] SEQ ID NO: 1417 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target FGF 18.NLRC4 Targeting with MG119-28

[0057] SEQ ID NO: 1418 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target NLRC4.NUDT16 Targeting with MG119-28

[0058] SEQ ID NO: 1419 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target NUDT16.EMC6 Targetingwith MG119-28

[0059] SEQ ID NO: 1420 shows the nucleotide sequence of sgRNA engineered to function with an MG119-28 nuclease to target EMC6.Valosin Containing Protein (VCP) Targetingwith MG119-28

[0060] SEQ ID NO: 1387 shows the nucleotide sequence of an sgRNA engineered to function with an MG119-28 nuclease in order to target a VCP gene.Nuclear Localization Signals

[0061] SEQ ID NOs: 369-384 and 1174-1203 show the amino acid amino acid sequences of nuclear localization signals.Gene Targeting SitesT Cell Receptor Alpha Constant (TRAC)

[0062] SEQ ID NOs: 505-536 show the DNA sequences of TRAC target sites.Apolipoprotein Al (APO Al)

[0063] SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036 show the DNA sequences of APOA1 target sites.APOA 1 Targeting with MG119-32

[0064] SEQ ID NOs: 1030-1036 show the DNA sequences of APOA1 target sites.- 9 - #592931Attorney Docket No : 00010.036.1801Albumin (ALB)

[0065] SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207 show the DNA sequences of albumin gene target sites.Albumin (ALB) Targeting with MG 119-28

[0066] SEQ ID NOs: 839-887. 1167, 1169, 1171, 1173. 1206, and 1207 show the DNA sequences of albumin gene target sites.Adeno-Associated Virus Integration Site 1 (AAVS1)

[0067] SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405 show the DNA sequences of AAVS1 target sites.A A VS 1 targeting w i th MG119-28

[0068] SEQ ID NO: 960-999, 1109-1135, and 1405 shows the nucleotide sequence of AAVS1 target site.AAVS1 Targeting with MG119-32

[0069] SEQ ID NOs: 1037-1046 show the DNA sequences of AAVS1 target sites.Vascular Endothelial Growth Factor A (VEGFA) Targeting with MG119-28

[0070] SEQ ID NO: 1406 shows the nucleotide sequence of a target site for human VEGFA. Interferon Gamma (IFNG) Targeting with MG 119-28

[0071] SEQ ID NO: 1407 shows the nucleotide sequence of a target site for human IFNG.Chloride Intracellular Channel 4 (CLIC4) Targeting with MG119-28

[0072] SEQ ID NO: 1408 shows the nucleotide sequence of a target site for human CLIC4. Fibroblast Growth Factor 18 (FGF18) Targeting with MG119-28

[0073] SEQ ID NO: 1409 shows the nucleotide sequence of atarget site for human FGF18. NLR Family CARD Domain Containing 4 (NLRC4) Targeting with MG 119-28

[0074] SEQ ID NO: 1410 shows the nucleotide sequence of a target site for human NLRC4. Nudix Hydrolase 16 (NUDT16) Targeting with MG119-28

[0075] SEQ ID NO: 1411 shows the nucleotide sequence of a target site for human NUDT16. ER Membrane Protein Complex Subunit 6 (EMC6) Targeting with MG119-28

[0076] SEQ ID NO: 1412 shows the nucleotide sequence of a target site for human EMC6.Valosin Containing Protein (VCP) Targeting

[0077] SEQ ID NO: 1391 shows the nucleotide sequence of a human VCP locus and SEQ ID NO:1392 shows the nucleotide sequence of mutant VCP locus from a human IBMPFD subject.

[0078] SEQ ID NO: 1393 shows the nucleotide sequence of a mouse VCP locus. SEQ ID NO: 1394 shows the nucleotide sequence of a mouse R155H / + strain-mutant VCP locus from a IBMPFD disease model mouse.- 10 - #592931Attorney Docket No : 00010.036.1801

[0079] SEQ ID NO: 1385 shows the DNA sequences of a VCP gene target site.

[0080] SEQ ID NO: 1386 shows the nucleotide sequence of a spacer sequence Cas9 sgRNA targeting mutant VCP locus.

[0081] SEQ ID NO: 1388 shows the nucleotide sequence of an sgRNA engineered to function with Cas9 in order to target a VCP gene.

[0082] SEQ ID NO: 1389 shows the nucleotide sequence of MG119-28 mRNA, designated as mr8.

[0083] SEQ ID NO: 1390 shows the nucleotide sequence of Cas9 mRNA, designated as mrl5.Ataxin 2 (ATXN2)

[0084] SEQ ID Nos: 1395-1397 show the nucleotide sequence of AAV cargo plasmid. SEQ ID NO: 1395 shows the nucleotide sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mATXN2-PlD6. SEQ ID NO: 1396 shows the nucleotide sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide mATXN2-PlC5. SEQ ID NO: 1397 shows the nucleotide sequence of AAV cargo plasmid expressing V5-tagged MG119-28 and guide R26g4.

[0085] SEQ ID NO: 1399 and 1401 show the nucleotide sequences of MG119-28 spacer sequence targeting mouse ATXN2.

[0086] SEQ ID NO: 1400 and 1402 show the nucleotide sequences of sgRNA engineered to function with MG119-28 in order to target a mouse ATXN2.

[0087] SEQ ID NO: 1403 shows the nucleotide sequence of an MG119-28 spacer sequence targeting mouse ROSA26.

[0088] SEQ ID NO: 1404 shows the nucleotide sequence of sgRNA engineered to function with MG119-28 in order to target a mouse ROSA26.OsCasllf

[0089] SEQ ID NO: 1384 shows the amino acid sequence of OsCasl2f nuclease.

[0090] SEQ ID NO: 1421 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl2f nuclease to target AAVS1.

[0091] SEQ ID NO: 1422 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl 2f nuclease to target VEGFA.

[0092] SEQ ID NO: 1423 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl 2f nuclease to target IFNG.

[0093] SEQ ID NO: 1424 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl 2f nuclease to target CLIC4.

[0094] SEQ ID NO: 1425 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl2f nuclease to target FGF18.- 11 - #592931Attorney Docket No : 00010.036.1801

[0095] SEQ ID NO: 1426 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl2f nuclease to target NLRC4.

[0096] SEQ ID NO: 1427 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl2f nuclease to target NUDT16.

[0097] SEQ ID NO: 1428 shows the nucleotide sequence of sgRNA engineered to function with an OsCasl 2f nuclease to target EMC6.LbCasl2a

[0098] SEQ ID NO: 1383 shows the amino acid sequence of LbCas 12a nuclease.

[0099] SEQ ID NO: 1429 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target AAV S 1.

[0100] SEQ ID NO: 1430 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target VEGFA.

[0101] SEQ ID NO: 1431 shows the nucleotide sequence of sgRNA engineered to function with an LbCasl2a nuclease to target IFNG.

[0102] SEQ ID NO: 1432 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target CLIC4.

[0103] SEQ ID NO: 1433 shows the nucleotide sequence of sgRNA engineered to function with an LbCasl2a nuclease to target FGF18.

[0104] SEQ ID NO: 1434 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target NLRC4.

[0105] SEQ ID NO: 1435 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target NUDT16.

[0106] SEQ ID NO: 1436 shows the nucleotide sequence of sgRNA engineered to function with an LbCas 12a nuclease to target EMC6.DETAILED DESCRIPTION

[0107] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by w ay of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[0108] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical - 12 - #592931Attorney Docket No : 00010.036.1801values. 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.

[0109] The practice of some methods disclosed herein employ, unless otherwise indicated, techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA.

[0110] As used herein, the singular forms '‘a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0111] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, w hich will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1 % of a given value.

[0112] The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotides are monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [aS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein encompasses dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of ddNTPs include, but are not limited to, ddATP, ddCTP. ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled, such as using moieties comprising optically detectable moi eties (e.g., fluorophores) or quantum dots. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzy me labels. Fluorescent labels of nucleotides include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue,- 13 - #592931Attorney Docket No : 00010.036.1801Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP. [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City. Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein-15-dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein- 12-UTP, and Fluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. The term nucleotide encompasses chemically modified nucleotides. An exemplary7chemically -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).

[0113] The terms ’‘polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multistranded form. Contemplated polynucleotides include a gene or fragment thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA). ribosomal RNA (rRNA), short interfering RNA (siRNA), shorthairpin RNA (shRNA), micro-RNA (miRNA), ribozymes. cDNA. recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA. A polynucleotide can be exogenous or endogenous to a cell and / or exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure are imparted before or after assembly of the polymer. Non-limiting examples of modifications include: 5-bromouracil, peptide nucleic acid, xeno nucleic - 14 - #592931Attorney Docket No : 00010.036.1801acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The sequence of nucleotides may be interrupted by nonnucleotide components.

[0114] The terms “peptide,'’ “polypeptide,” and “protein'’ are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, refer to natural and non-natural amino acids, including, but not limited to, modified amino acids. Modified amino acids include amino acids that have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. The term “amino acid” includes both D-amino acids and L-amino acids.

[0115] As used herein, “operably linked”, “operable linkage”, “operatively linked”, or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein an operation (e.g., movement or activation) of a first genetic element has some effect on the second genetic element. The effect on the second genetic element can be, but need not be, of the same type as operation of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes an activation of the second element. For instance, a regulatory element, which may comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0116] The terms '‘transfection” or '‘transfected” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof.- 15 - #592931Attorney Docket No : 00010.036.1801

[0117] As used herein, the “non-native” refers to a nucleic acid or polypeptide sequence that is non-naturally occurring. Non-native refers to a non-naturally occurring nucleic acid or polypeptide sequence that comprises modifications such as mutations, insertions, or deletions. The term non-native encompasses fusion nucleic acids or polypeptides that encodes or exhibits an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) of the nucleic acid or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence includes those linked to a naturally-occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence encoding a chimeric nucleic acid or polypeptide.

[0118] The term “promoter”, as used herein, refers to the regulatory DNA region which controls transcription or expression of a polynucleotide (e.g., a gene) and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. Eukary otic basal promoters ty pically, though not necessarily, contain a TATA-box and / or a CAAT box.

[0119] The term “expression”, as used herein, refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, the term expression includes splicing of the mRNA in a eukaryotic cell.

[0120] A “vector” as used herein, refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which mediates delivery7of the polynucleotide to a cell. Examples of vectors include nucleic-based vectors (e.g., plasmids and viral vectors) and liposomes. An exemplary nucleic-acid based vector comprises genetic elements, e.g.. regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0121] As used herein, “expression cassette” and “nucleic acid cassette” are used interchangeably to refer to a component of a vector comprising a combination of nucleic acid sequences or elements (e.g., therapeutic gene, promoter, and a terminator) that are expressed together or are operably linked for expression. The terms encompass an expression cassette including a combination of regulatory elements and a gene or genes to which they are operably7linked for expression.- 16 - #592931Attorney Docket No : 00010.036.1801

[0122] A "functional fragment” of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) that is substantially similar to a biological activity of the full-length DNA or protein sequence. A biological activity7of a DNA sequence includes its ability to influence expression in a manner attributed to the full-length sequence.

[0123] The terms "engineered.” “synthetic,” and “artificial” are used interchangeably herein to refer to an object that has been modified by human intervention. For example, the terms refer to a polynucleotide or polypeptide that is non-naturally occurring. An engineered peptide has, but does not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity7, less than 1% sequence identity ) to a naturally occurring human protein. For example, VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: a nucleic acid modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid synthesized in vitro with a sequence that does not exist in nature; a protein modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein acquiring anew function or property. An “engineered” system comprises at least one engineered component.

[0124] As used herein, a “guide nucleic acid” or “guide polynucleotide” refers to a nucleic acid that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid can include a crRNA or a tracrRNA or a combination of both. The term guide nucleic acid encompasses an engineered guide nucleic acid and a programmable guide nucleic acid to specifically bind to the target nucleic acid. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary7to and hybridizes with the guide nucleic acid is the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore is not complementary to the guide nucleic acid is called noncomplementary strand. A guide nucleic acid having a polynucleotide chain is a “single guide nucleic acid.” A guide nucleic acid having two polynucleotide chains is a “double guide nucleic acid.” If not otherwise specified, the term “guide nucleic acid” is inclusive, referring to both single guide nucleic acids and double guide nucleic acids. A guide nucleic acid may comprise a segment referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence,” or a “spacer.” A nucleic acid-targeting segment- 17 - #592931Attorney Docket No : 00010.036.1801can include a sub-segment referred to as a "protein binding segment’’ or ‘'protein binding sequence” or "Cas protein binding segment.”

[0125] As used herein, the term “Casl2a” refers to a family of Cas endonucleases that are class 2, Type V-A Cas endonucleases and that (a) use a relatively small guide RNA (about 42-44 nucleotides) that is processed by the nuclease itself following transcription from the CRISPR array, and (b) cleave DNA to leave staggered cut sites. Further features of this family of enzymes can be found, e.g. in Zetsche B, Heidenreich M, Mohanraju P, et al. Nat Biotechnol 2017;35:31-34, and Zetsche B, Gootenberg JS, Abudayyeh 00, et al. Cell 2015;163:759-771.

[0126] The term “tracrRNA” or “tracr sequence” means trans-activating CRISPR RNA. tracrRNA interacts with the CRISPR (cr) RNA to form a guide nucleic acid (e.g., guide RNA or gRNA) that may hybridize to a target nucleic acid and thereby directs an associated nuclease to the target nucleic acid.

[0127] As used herein, the term “complex” refers to a joining of at least two components. The two components may each retain the properties / activities they had prior to forming the complex or gain properties as a result of forming the complex. The joining includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, Van der Waals interactions, and hydrophobic bond), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, a complex comprises an endonuclease and a guide polynucleotide.

[0128] The term “sequence identity” or “percent identity” in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, e.g., BLASTP using parameters of a wordlength (W) of 3. an expectation (E) of 10, and the BLOSUM62 scoring matrix setting gap costs at existence of 11. extension of 1, and using a conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and the PAM30 scoring matrix setting gap costs at 9 to open gaps and 1 to extend gaps for sequences of less than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW with the Smith-Waterman homology search algorithm parameters with a match of 2, a mismatch of -1, and a gap of -1; MUSCLE with default- 18 - #592931Attorney Docket No : 00010.036.1801parameters; MAFFT with parameters of a retree of 2 and max iterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0129] The term “optimally aligned’' in the context of two or more nucleic acids or polypeptide sequences, refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that have been aligned to maximal correspondence of amino acids residues or nucleotides, for example, as determined by the alignment producing a highest or “optimized” percent identity score.

[0130] Included in the current disclosure are variants of any of the enzy mes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%. at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the endonuclease protein sequences described herein (e.g. MG90, MG91A, MG91B. MG91C. MG118, MG119, MG120, MG122, MG126, or MG191 family endonucleases described herein, or any other family nuclease described herein). In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of one or more critical active site residues or guide RNA binding residues of the endonuclease are not disrupted. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of at least one of the conserved or functional residues. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of all of the conserved or functional residues.

[0131] Also included in the current disclosure are variants of any of the enzymes described herein with substitution of one or more catalytic residues to decrease or eliminate activity of the enzyme (e.g. decreased-activity variants). In some embodiments, a decreased activity variant as a protein- 19 - #592931Attorney Docket No : 00010.036.1801described herein comprises a disrupting substitution of at least one, at least two, or all three catalytic residues.

[0132] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see. for e.g., Creighton. Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)Overview

[0133] The discovery of new Cas enzymes with unique functionality and structure offers the potential to further gene editing technologies, improving speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems in microbes and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is partly because a huge number of microbial species may not be readily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches containing large numbers of microbial species may offer the potential to drastically increase the number of new CRISPR / Cas systems characterized and speed the discovery of new7oligonucleotide editing functionalities. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of CasX / CasY CRISPR systems from metagenomic analysis of natural microbial communities.

[0134] CRISPR / Cas systems are RNA-directed nuclease complexes that function as an adaptive immune system in microbes. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally are made up of two parts: (i) an array of short repetitive sequences (30-40 bp) separated by short spacer sequences, which encode the RNA-based targeting element; and (ii) ORFs encoding the Cas nuclease. Efficient nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary7hybridization betw een the first 6-8 nucleic acids of the target nucleic acid - 20 - #592931Attorney Docket No : 00010.036.1801and a crRNA guide; and (ii) presence of a protospacer- adjacent motif (PAM) sequence within a certain vicinity of the target nucleic acid sequence depending on the specific Cas nuclease (the PAM usually being a sequence not commonly represented within the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are commonly organized into 2 classes, 5 types and 16 subtvpes based on shared functional characteristics and evolutionary similarity.

[0135] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes, and include Types I, III, and IV Cas nucleases. Class 2 CRISPR-Cas systems generally have single-polypeptide multidomain nuclease effectors, and include Types II. V and VI Cas nucleases.

[0136] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, the processing of the CRISPR array into mature crRNAs does not require the presence of a special endonuclease subunit, but rather a small trans-encoded crRNA (tracrRNA) with a region complementary to the array repeat sequence; the tracrRNA interacts with both its corresponding effector nuclease (e.g. Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are identified as DNA nucleases. Type 2 effectors generally exhibit a structure comprising a RuvC-like endonuclease domain that adopts the RNase H fold with an unrelated HNH nuclease domain inserted within the folds of the RuvC-like nuclease domain. The RuvC-like domain is responsible for the cleavage of the target (e.g., crRNA complementary') DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0137] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g. Cas 12) structure similar to that of Type II effectors, comprising a RuvC-like domain. Similar to Type II, most (but not all) Type V CRISPR systems use a tracrRNA to process pre-crRNAs into mature crRNAs. However, unlike Type II systems which requires RNAse III to cleave the pre-crRNA into multiple crRNAs. Type V systems are capable of using the effector nuclease itself to cleave pre-crRNAs. Like Type-II CRISPR-Cas systems, Type V CRISPR-Cas systems are again identified as DNA nucleases. Unlike Type II CRISPR-Cas systems, some Type V enzymes (e g., Cas 12a) appear to have a robust single-stranded nonspecific deoxyribonuclease activity that is activated by the first crRNA directed cleavage of a double-stranded target sequence.

[0138] CRISPR-Cas systems have emerged in recent years as the gene editing technology of choice due to their targetability and ease of use. The most commonly used systems are the Class 2 Type II SpCas9 and the Class 2 Type V-A Casl2a (previously Cpfl). The Type V-A systems in particular are becoming more widely used since their reported specificity in cells is higher than - 21 - #592931Attorney Docket No : 00010.036.1801other nucleases, with fewer or no off-target effects. The V -A systems are also advantageous in that the guide RNA is small (42-44 nucleotides compared with approximately 100 nt for SpCas9) and is processed by the nuclease itself following transcription from the CRISPR array, simplifying multiplexed applications with multiple gene edits. Furthermore, the V-A systems have staggered cut sites, which may facilitate directed repair pathways, such as microhomology-dependent targeted integration (MITI).

[0139] The most commonly used Type V-A enzymes require a 5‘ protospacer adjacent motif (PAM) next to the chosen target site: 5’-TTTV-3' for Lachnospiraceae bacterium ND2006 LbCasl2a and Acidammococcus sp. AsCasl2a; and 5’-TTV-3' for Francisella novicidaFnCasl2a. Recent exploration of orthologs has revealed proteins with less restrictive PAM sequences that are also active in mammalian cell culture, for example YTV, YYN or TTN. However, these enzymes do not fully encompass Type V biodiversify and targetabilify, and may not represent all possible activities and PAM sequence requirements. Described herein, in certain embodiments, are improved Type V nucleases that are highly targetable, compact, and precise for use in systems and methods for gene editing.MG Enzymes

[0140] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease and an engineered guide polynucleotide. In some embodiments, the endonuclease is a Class 2, Type V endonuclease. In some embodiments, the endonuclease is a double-strand nuclease. In some embodiments, the endonuclease is catalytically dead. In some embodiments, the endonuclease is modified. In some embodiments, the endonuclease is modified resulting in an endonuclease with nickase activity. In some embodiments, the modified endonuclease is a site-directed nickase.

[0141] In some embodiments, the endonuclease is an MG119 endonuclease.

[0142] In some embodiments, the nucleases are less than about 1000 amino acids in length. In some embodiments, the nucleases are less than about 900 amino acids in length. In some embodiments, the nucleases are less than about 850 amino acids in length. In some embodiments, the nucleases are less than about 800 amino acids in length. In some embodiments, the nucleases are less than about 750 amino acids in length. In some embodiments, the nucleases are less than about 700 amino acids in length. In some embodiments, the nucleases are less than about 650 amino acids in length. In some embodiments, the nucleases are less than about 600 amino acids in length. In some embodiments, the nucleases are less than about 550 amino acids in length. In some embodiments, the nucleases are less than about 500 amino acids in length.- 22 - #592931Attorney Docket No : 00010.036.1801

[0143] In some embodiments, the endonuclease is a MG119 endonuclease (i. e. , SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382). In some embodiments, the endonuclease has at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%. at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 70% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 75% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 80% identity’ to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 85% identity’ to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 90% identity to any one of SEQ ID NOs: 1-121, 182-193. 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 95% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 96% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 97% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 98% identity’ to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has at least about 99% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382. In some embodiments, the endonuclease has 100% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382.

[0144] In some embodiments, the endonuclease has 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%. at least about 99% or 100% identity to SEQ ID NO: 28. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 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%, at least about 99% or 100% sequence identity to SEQ ID NO: 1382. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 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 - 23 - #592931Attorney Docket No : 00010.036.180185%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 1382.

[0145] In some embodiments, the endonuclease comprises one or more amino acid modifications compared to a MG119 endonuclease described herein. In some embodiments, the endonuclease comprises one or more amino acid modifications as compared to SEQ ID NO: 28 (i.e.. MG119-28). In some embodiments, the endonuclease comprises one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 28.

[0146] In some embodiments, the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification K93R as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification E202K. as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification M204K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modification N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, M204K, and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E236K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, E236K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K, E236K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, M204K, and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K and E236K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E236K and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K.93R, E236K, and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E236K and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and N329E as - 24 - #592931Attorney Docket No : 00010.036.1801compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, M204K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R and E202K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, E236K, and E255K, as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E236K, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R and M204K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, M204K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E255K and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and M204K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K and M204K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, E202K, and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K, E236K, and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K93R, M204K, and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications M204K, E255K, and N329E as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications E202K, M204K, and E255K as compared to SEQ ID NO: 28. In some embodiments, the endonuclease comprises the modifications K.93R, E202K, M204K, E236K, E255K, andN329E as compared to SEQ ID NO: 28.- 25 - #592931Attorney Docket No : 00010.036.1801

[0147] In some embodiments, the nucleases comprise RuvC catalytic residues. In some embodiments, the nucleases do not require tracrRNA. In some embodiments, the nucleases comprise RuvC catalytic residues and do not require tracrRNA.

[0148] In some embodiments, the nucleases comprise a PAM interacting domain.

[0149] In some embodiments, the engineered nuclease system is discovered through metagenomic sequencing. In some embodiments, the metagenomic sequencing is conducted on samples collected from various environments. In some embodiments, the environment is a human microbiome, an animal microbiome, an environment with high temperatures, an environment with low temperatures, or sediment.

[0150] In some embodiments, the endonuclease comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is at an N-terminus of the endonuclease. In some embodiments, the NLS is at a C-terminus of the endonuclease. In some embodiments, the NLS is at an N-terminus and a C-terminus of the endonuclease.

[0151] In some embodiments, the NLS comprises a sequence of any one of SEQ ID NOs: 369-384 and 1174-1203, or a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%. at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%. at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 80% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 85% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 90% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 91% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 92% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 93% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 94% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 95% identity to SEQ ID NOs: 369-384 and 1 174-1203. In some cases, the NLS has at least about 96% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 97% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 98% identity to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has at least about 99% identity’ to SEQ ID NOs: 369-384 and 1174-1203. In some cases, the NLS has 100% identity to SEQ ID NOs: 369-384 and 1174-1203.- 26 - #592931Attorney Docket No : 00010.036.1801Table 1: Example NLS Sequences that may be used with Cas effectors according to the disclosure.<<<<<<- 27 - #592931Attorney Docket No : 00010.036.1801

[0152] In some cases, the engineered nuclease system further comprises a single- or doublestranded DNA repair template. In some cases, the engineered nuclease system further comprises a single-stranded DNA repair template.

[0153] In some cases, the single- or double-stranded DNA repair template comprises from 5’ to 3’: a first homology' arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3' to the target sequence.

[0154] In some cases, the first homology arm comprises a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides. In some cases, the second homology arm comprises a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, or at least 1000 nucleotides.

[0155] In some cases, the first and second homology arms are homologous to a genomic sequence of a prokaryote. In some cases, the first and second homology’ arms are homologous to a genomic sequence of a bacteria. In some cases, the first and second homology arms are homologous to a genomic sequence of a fungus. In some cases, the first and second homology- arms are homologous to a genomic sequence of a eukaryote.

[0156] In some cases, the engineered nuclease system further comprises a DNA repair template comprising a double-stranded DNA segment flanked by one or two single-stranded DNA - 28 - #592931Attorney Docket No : 00010.036.1801segments. In some embodiments, the single-stranded DNA segments are conjugated to the 5' ends of the double-stranded DNA segment. In some embodiments, the double-stranded DNA segment is flanked by two single-stranded DNA segments. In some embodiments, the single-stranded DNA segments are conjugated to the 3' ends of the double-stranded DNA segment. In some embodiments, the -stranded DNA segments have a length from 4 to 10 nucleotide bases. In some embodiments, the single-stranded DNA segments have a nucleotide sequence complementary to a sequence within the spacer sequence.

[0157] In some cases, the single-stranded DNA segments have a length from 1 to 15 nucleotide bases. In some cases, the single-stranded DNA segments have a length from 4 to 10 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 4 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 5 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 6 nucleotide bases. In some cases, the singlestranded DNA segments have a length of 7 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 8 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 9 nucleotide bases. In some cases, the single-stranded DNA segments have a length of 10 nucleotide bases.

[0158] In some cases, the single-stranded DNA segments have a nucleotide sequence complementary to a sequence within the spacer sequence. In some cases, the double-stranded DNA sequence comprises a barcode, an open reading frame, an enhancer, a promoter, a protein-coding sequence, a miRNA coding sequence, an RNA coding sequence, or a transgene. In some embodiments, the double-stranded DNA sequence is flanked by a nuclease cut site. In some embodiments, the nuclease cut site comprises a spacer and a PAM sequence.

[0159] In some cases, the engineered nuclease system further comprises a source of Mg2+.

[0160] In some cases, the sequence is determined by a BLASTP, CLUSTALW, MUSCLE, or MAFFT algorithm, or a CLUSTALW algorithm with the Smith-Waterman homology7search algorithm parameters. In some cases, the sequence is determined by the BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.Guide Polynucleotides

[0161] In some embodiments, the engineered nuclease system disclosed herein comprises an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or a dual guide RNA.- 29 - #592931Attorney Docket No : 00010.036.1801

[0162] In some embodiments, the engineered polynucleotide has at least about 70% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 75% identity to any one of SEQ ID NOs: 122-155. 194-215. 385-504. 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 80% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165. 1209-1293, 1387, 1400. 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 85% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 90% identity to any one of SEQ ID NOs: 122-155. 194-215.385-504.790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 95% identity to any one of SEQ ID NOs: 122-155. 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165. 1209-1293, 1387, 1400. 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 96% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 97% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 98% identity to any one of SEQ ID NOs: 122-155. 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165. 1209-1293, 1387, 1400. 1402, 1404. 1413-1420, 365-368, 1166, 1 168, 1170, and 1172. In some embodiments, the engineered polynucleotide has at least about 99% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400. 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the engineered polynucleotide comprises 100% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, 1387, 1400, 1402, 1404, 1413-1420, 365-368, 1166, 1168, 1170, and 1172.- 30 - #592931Attorney Docket No : 00010.036.1801

[0163] In some embodiments, the engineered polynucleotide has at least about 70% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 75% identity to any one of SEQ ID NOs: 201. 365-368. 424-449. 790-838. 888-903. 920-959. 1082-1108, 1136-1166, 1168, 1 170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 80% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245. 1246-1293, 1387, 1400, 1402. 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 85% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 90% identity to any one of SEQ ID NOs: 201, 365-368. 424-449. 790-838. 888-903. 920-959, 1082-1108, 1136-1166. 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 95% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903. 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245. 1246-1293, 1387, 1400, 1402. 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 96% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 97% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 98% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903. 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245. 1246-1293, 1387, 1400, 1402. 1404. 1413, and 1414-1420. In some embodiments, the engineered polynucleotide has at least about 99% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420. In some embodiments, the engineered polynucleotide comprises 100% identity to any one of SEQ ID NOs: 201, 365-368, 424-449, 790-838, 888-903, 920-959, 1082-1108, 1136-1166, 1168, 1170, 1172, 1212-1245, 1246-1293, 1387, 1400, 1402, 1404, 1413, and 1414-1420.- 31 - #592931Attorney Docket No : 00010.036.1801

[0164] In some embodiments, the engineered guide polynucleotide targets a gene in a cell. In some embodiments, the engineered guide polynucleotide targets a gene in a mammalian cell. In some embodiments, the mammalian cell is a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, or a human cell. In some embodiments, the target gene or target locus is albumin, T cell receptor alpha constant (TRAC), adeno-associated virus integration site 1 (AAVS1), Ataxin 2 (ATXN2), Apolipoprotein Al (APOA1), superoxide dismutase 1 (SOD1), Valosin containing protein (VCP), vascular endothelial growth factor A (VEGFA), interferon gamma (IFNG), chloride intracellular channel 4 (CLIC4), fibroblast growth factor 18 (FGF18), NLR family CARD domain containing 4 (NLRC4), nudix hydrolase 16 (NUDT16), or ER membrane protein complex subunit 6 (EMC6).

[0165] In some embodiments, the target gene is ATXN2. In some embodiments, the guide polynucleotide targeting ATXN2 is encoded by any one of SEQ ID NOs: 1400, 1402, and 1404, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1400. 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1400, 1402. and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity' to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1400, 1402, and 1404. In some embodiments, the guide - 32 - #592931Attorney Docket No : 00010.036.1801polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1400, 1402, and 1404.

[0166] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the ATXN2 gene or within an intron of the ATXN2 gene (e.g.. SEQ ID NOs: 1399, 1401, or 1403). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 1399, 1401 , and 1403 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1399, 1401, and 1403. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 1399, 1401, and 1403.

[0167] In some embodiments, the target gene is TRAC. In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TRAC gene or within an intron of the TRAC gene (e.g., SEQ ID NOs: 505-536). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 505-536 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95%- 33 - #592931Attorney Docket No : 00010.036.1801identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 505-536. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 505-536.

[0168] In some embodiments, the target gene is APOA1. In some embodiments, the guide polynucleotide targeting APOA1 is encoded by any one of SEQ ID NOs: 888-903 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%. at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity' to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 888-903.- 34 - #592931Attorney Docket No : 00010.036.1801

[0169] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the APOA1 gene or within an intron of the APOA1 gene (e.g., SEQ ID NOs: 888-903). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 888-903 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 80% identity' to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a sequence having at least about 85% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to any' one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a sequence having at least about 95% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 96% identity to any' one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 97% identity' to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 98% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 99% identity' to any one of SEQ ID NOs: 888-903. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having 100% identity to any one of SEQ ID NOs: 888-903.

[0170] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the APOA1 gene or within an intron of the APOA1 gene (e.g., SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity' to any one of SEQ ID NOs: 537-653, 703-721. 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity' to any one of SEQ ID NOs:- 35 - #592931Attorney Docket No : 00010.036.1801537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 537-653. 703-721. 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity' to any one of SEQ ID NOs: 537-653, 703-721. 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity' to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036.

[0171] In some embodiments, the target gene is AAVS1. In some embodiments, the guide polynucleotide targeting AAVS1 is encoded by any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165. and 1212-1245, and 1413. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 920-959, 1082-1108,- 36 - #592931Attorney Docket No : 00010.036.18011136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413.

[0172] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the AAVS 1 gene or within an intron of the AAVS1 gene (e.g., SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to any one of SEQ ID NOs : 920-959. 1082- 1108. 1136- 1165 , and 1212- 1245 , and 1413 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 97% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence - 37 - #592931Attorney Docket No : 00010.036.1801complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413.

[0173] In some embodiments, the guide polynucleotide targeting AAVS1 is encoded by any one of SEQ ID NOs: 202 and 450-466, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity' to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 202 and 450-466. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 202 and 450-466.

[0174] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the AAVS1 gene or within an intron of the AAVS1 gene (e.g.. SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to - 38 - #592931Attorney Docket No : 00010.036.1801any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity’ to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135. and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity’ to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135. and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity’ to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity’ to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405.

[0175] In some embodiments, the target gene is superoxide dismutase 1 (SOD1). In some embodiments, the guide polynucleotide targeting SOD1 is encoded by any one of SEQ ID NOs: 1246-1293, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity7to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity' to any one of SEQ ID NOs: 1246-1293. In some embodiments, the - 39 - #592931Attorney Docket No : 00010.036.1801guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1257-1324, 1523-1562, and 1677-1686. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1246-1293.

[0176] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a target nucleic acid sequence within the SOD1 gene or within an intron of the SOD1 gene (e g., SEQ ID NOs: 1246-1293). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 1246-1293 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having 100% identity to any one of SEQ ID NOs: 1246-1293.- 40 - #592931Attorney Docket No : 00010.036.1801

[0177] In some embodiments, the target gene is albumin. In some embodiments, the guide polynucleotide targeting albumin is encoded by any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 790-838. 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one ofSEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and ll72. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 790-838, 365-368. 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity’ to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity’ to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 790-838. 365-368. 1166, 1168. 1170, and 1172. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity’ to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172.

[0178] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a target nucleic acid sequence within the albumin gene or within an intron of the albumin gene (e.g., SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one ofSEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172 or a sequence having at least 90%,- 41 - #592931Attorney Docket No : 00010.036.180195%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity7to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 98% identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170. and 1172. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to any one ofSEQ ID NOs: 790-838, 365-368, 1166, 1168, 1170, and ll72. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 790-838. 365-368. 1166, 1168, 1170, and 1172.

[0179] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the albumin gene or within an intron of the albumin gene (e.g., SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In - 42 - #592931Attorney Docket No : 00010.036.1801some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ IDNOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206. and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029. 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 654-702, 839-887. 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity' to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207.

[0180] In some embodiments, the target gene is valosin containing protein (VCP). In some embodiments, the guide polynucleotide targeting VCP is encoded by SEQ ID NO: 1387. or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity' to SEQ ID NO: 1387 In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 138776. In some embodiments, the - 43 - #592931Attorney Docket No : 00010.036.1801guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity7to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1387.

[0181] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence w ithin the VCP gene or within an intron of the VCP gene (e.g., SEQ ID NO: 1387). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1387or a sequence having at least 90%, 95%, 97%. 98%. or 99% sequence identity’ to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity^ to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity7to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary7to a sequence having at least about 90% identity7to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary7to a sequence having at least about 95% identity7to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary- to a sequence having at least about 98% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to SEQ ID NO: 1387. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity7to SEQ ID NO: 1387.

[0182] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the VCP gene or within an intron of the VCP gene (e.g., SEQ ID NO: 1385, 1391. 1392. 1393, or 1394). In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity- to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity7to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or - 44 - #592931Attorney Docket No : 00010.036.1801targets a sequence having at least about 90% identity to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity7to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to SEQ ID NO: 1385, 1391, 1392. 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity7to SEQ ID NO: 1385, 1391, 1392, 1393, or 1394.

[0183] In some embodiments, the target gene is vascular endothelial growth factor A (VEGFA). In some embodiments, the guide polynucleotide targeting VEGFA is encoded by SEQ ID NO: 1414 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%. at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity7to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity7to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity7to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1414. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1414.- 45 - #592931Attorney Docket No : 00010.036.1801

[0184] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the VEGFA gene or within an intron of the VEGFA gene (e.g., SEQ ID NO: 1406). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1406 or a sequence having at least 90%. 95%. 97%. 98%. or 99% sequence identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 85% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 97% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 98% identity to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity’ to SEQ ID NO: 1406. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having 100% identity to SEQ ID NO: 1406.

[0185] In some embodiments, the target gene is Interferon Gamma (IFNG). In some embodiments, the guide polynucleotide targeting IFNG is encoded by SEQ ID NO: 1415 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a - 46 - #592931Attorney Docket No : 00010.036.1801sequence having at least about 90% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1415. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1415.

[0186] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the IFNG gene or within an intron of the IFNG gene (e.g., SEQ ID NO: 1407). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1407 or a sequence having at least 90%, 95%, 97%, 98%. or 99% sequence identity' to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity' to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity’ to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to SEQ ID NO: 1407. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to SEQ ID NO: 1407.

[0187] In some embodiments, the target gene is Chloride intracellular channel 4 (CLIC4). In some embodiments, the guide polynucleotide targeting CLIC4 is encoded by SEQ ID NO: 1416 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46- - 47 - #592931Attorney Docket No : 00010.036.180180 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%. at least about 96%. at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity7to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1416. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1416.

[0188] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the CLIC4 gene or within an intron of the CLIC4 gene (e g., SEQ ID NO: 1408). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1408 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity' to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity' to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence - 48 - #592931Attorney Docket No : 00010.036.1801complementary to a sequence having at least about 98% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to SEQ ID NO: 1408. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to SEQ ID NO: 1408.

[0189] In some embodiments, the target gene is Fibroblast Growth Factor 18 (FGF18). In some embodiments, the guide polynucleotide targeting FGF18 is encoded by SEQ ID NO: 1417 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity7to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1417. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1417.

[0190] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary7to a target nucleic acid sequence within the FGF18 gene or within an intron of the FGF18 gene (e g., SEQ ID NO: 1409). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1409 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or - 49 - #592931Attorney Docket No : 00010.036.1801targets a sequence complementary to a sequence having at least about 85% identity to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 90% identity to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity7to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity’ to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity’ to SEQ ID NO: 1409. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to SEQ ID NO: 1409.

[0191] In some embodiments, the target gene is NLR family CARD domain-containing protein 4 (NLRC4). In some embodiments, the guide polynucleotide targeting NLRC4 is encoded by SEQ ID NO: 1418 or a sequence having at least 90%. 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%. at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity’ to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity’ to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by - 50 - #592931Attorney Docket No : 00010.036.1801a sequence having at least about 99% identity to SEQ ID NO: 1418. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1418.

[0192] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the NLRC4 gene or within an intron of the NLRC4 gene (e.g.. SEQ ID NO: 1410). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1410 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity' to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 98% identity’ to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 99% identity to SEQ ID NO: 1410. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to SEQ ID NO: 1410.

[0193] In some embodiments, the target gene is nudix hydrolase 16 (NUDT16). In some embodiments, the guide polynucleotide targeting NUDT16 is encoded by SEQ ID NO: 1419 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1419. In some - 51 - #592931Attorney Docket No : 00010.036.1801embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1419. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1419.

[0194] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a target nucleic acid sequence within the NUDT16 gene or within an intron of the NUDT16 gene (e.g., SEQ ID NO: 1411). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 1411 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 85% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary^ to a sequence having at least about 96% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to SEQ ID NO: 1411. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a sequence having 100% identity to SEQ ID NO: 1411.

[0195] In some embodiments, the target gene is ER Membrane Protein Complex Subunit 6 (EMC6). In some embodiments, the guide polynucleotide targeting EMC6 is encoded by SEQ ID - 52 - #592931Attorney Docket No : 00010.036.1801NO: 1420 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 consecutive nucleotides having at least about 20%, at least about 25%, at least about 30%. at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%. at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 1420. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 1420.

[0196] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary’ to a target nucleic acid sequence within the EMC6 gene or within an intron of the EMC6 gene (e.g., SEQ ID NO: 1412). In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to SEQ ID NO: 1412 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity' to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 80% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 85% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary' to a sequence having at least about 90% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 95% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 96% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or - 53 - #592931Attorney Docket No : 00010.036.1801targets a sequence complementary to a sequence having at least about 97% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 98% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having at least about 99% identity to SEQ ID NO: 1412. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity’ to SEQ ID NO: 1412.

[0197] In some embodiments, the engineered guide polynucleotide is configured to form a complex with the endonuclease. In some cases, the engineered guide polynucleotide comprises a spacer sequence. In some cases, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some cases, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0198] In some embodiments, the engineered guide polynucleotide comprises a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target nucleic acid site; and a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex. In some embodiments, the two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides. In some embodiments, the engineered guide polynucleotide is capable of forming an endonuclease (e.g., a Class 2, Type V Cas endonuclease). In some embodiments, the DNA-targeting segment is positioned 3' of both of the two complementary stretches of nucleotides.

[0199] In some cases, the endonuclease is not a Cpfl or Cmsl endonuclease.

[0200] In some cases, the endonuclease is configured to bind to the engineered guide polynucleotide. In some cases, the Cas endonuclease is configured to bind to the engineered guide polynucleotide. In some cases, the class 2 Cas endonuclease is configured to bind to the engineered guide polynucleotide. In some cases, the class 2, type V Cas endonuclease is configured to bind to the engineered guide polynucleotide. In some cases, the class 2, type V, subtype Cas endonuclease is configured to bind to the engineered guide polynucleotide.

[0201] In some embodiments, the guide polynucleotide is configured to form a complex with the endonuclease. In some embodiments, the guide polynucleotide binds to the endonuclease to form a complex. In some embodiments, the guide polynucleotide binds (e.g., non-covalently through electrostatic interactions or hydrogen bonds) to the endonuclease to form a complex. In some embodiments, the guide polynucleotide is fused to the endonuclease to form a complex.

[0202] In some cases, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases,- 54 - #592931Attorney Docket No : 00010.036.1801the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary' to a fungal genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary' to a human genomic polynucleotide sequence.

[0203] In some cases, the guide polynucleotide comprises a hairpin comprising at least 8 basepaired ribonucleotides. In some cases, the guide polynucleotide comprises a hairpin comprising at least 9 base-paired ribonucleotides. In some cases, the guide polynucleotide comprises a hairpin comprising at least 10 base-paired ribonucleotides. In some cases, the guide polynucleotide comprises a hairpin comprising at least 11 base-paired ribonucleotides. In some cases, the guide polynucleotide comprises a hairpin comprising at least 12 base-paired ribonucleotides.

[0204] In some cases, the guide polynucleotide is 30-250 nucleotides in length. In some cases, the guide polynucleotide is 42-44 nucleotides in length. In some cases, the guide polynucleotide is 42 nucleotides in length. In some cases, the guide polynucleotide is 43 nucleotides in length. In some cases, the guide polynucleotide is 44 nucleotides in length. In some cases, the guide polynucleotide is 85-245 nucleotides in length. In some cases, the guide polynucleotide is more than 90 nucleotides in length. In some cases, the guide polynucleotide is less than 245 nucleotides in length.

[0205] In some embodiments, the guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide polynucleotide comprises one or more inter-nucleoside linkers modified from the natural phosphodiester. In some embodiments, all of the inter-nucleoside linkers of the guide polynucleotide, or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the inter nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.

[0206] In some embodiments, the present disclosure provides an engineered guide polynucleotide comprising a DNA-targeting segment. In some cases, the DNA-targeting segment comprises a nucleotide sequence that is complementary' to a target sequence. In some cases, the target sequence is in a target DNA molecule. In some cases, the engineered guide polynucleotide comprises a protein-binding segment. In some cases, the protein-binding segment comprises two complementary' stretches of nucleotides. In some cases, the two complementary stretches of nucleotides hybridize to form a double-stranded RNA (dsRNA) duplex. In some cases, the two complementary' stretches of nucleotides are covalently linked to one another with intervening - 55 - #592931Attorney Docket No : 00010.036.1801nucleotides. In some cases, the engineered guide ribonucleic acid polynucleotide is configured to form a complex with an endonuclease. In some cases, the complex targets the target sequence of the target DNA molecule. In some cases, the DNA-targeting segment is positioned 3‘ of both of the two complementary stretches of nucleotides.

[0207] In some cases, the double-stranded RNA (dsRNA) duplex comprises at least 8 ribonucleotides. In some cases, the double-stranded RNA (dsRNA) duplex comprises at least 9 ribonucleotides. In some cases, the double-stranded RNA (dsRNA) duplex comprises at least 10 ribonucleotides. In some cases, the double-stranded RNA (dsRNA) duplex comprises at least 11 ribonucleotides. In some cases, the double-stranded RNA (dsRNA) duplex comprises at least 12 ribonucleotides.

[0208] In some embodiments, the guide polynucleotide comprises modifications to a ribose sugar or nucleobase. In some embodiments, the guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the nbose sugar moiety found in deoxyribose nucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e g., UNA). In some embodiments, the sugar-modified nucleosides comprise bicyclohexose nucleic acids or tricyclic nucleic acids. In some embodiments, the modified nucleosides comprise nucleosides where the sugar moiety is replaced with a non-sugar moiety7, for example peptide nucleic acids (PNA) or morpholino nucleic acids.

[0209] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modifications comprise modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, substituents are introduced at the 2’, 3’, 4’, or 5’ positions, or combinations thereof. In some embodiments, nucleosides with modified sugar moieties comprise 2’ modified nucleosides, e.g., 2’ substituted nucleosides. A 2’ sugar modified nucleoside, in some embodiments, is a nucleoside that has a substituent other than -H or -OH at the 2‘ position (2' substituted nucleoside) or comprises a 2’ linked biradical, and comprises 2' substituted nucleosides and LNA (2'-4’ biradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides comprise, but are not limited to, 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleosides. In some embodiments, the modification in the ribose group comprises a - 56 - #592931Attorney Docket No : 00010.036.1801modification at the 2’ position of the ribose group. In some embodiments, the modification at the 2’ position of the ribose group is selected from the group consisting of 2’-O-methyl, 2’-fluoro, 2’-deoxy, and 2’-O-(2-methoxyethyl).

[0210] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In certain embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2’-O-methoxyethyl group. In some embodiments, the guide polynucleotide comprises both inter-nucleoside linker modifications and nucleoside modifications.

[0211] In some cases, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0212] In some cases, the guide polynucleotide is 30-400 nucleotides in length. In some cases, the guide polynucleotide is 85-245 nucleotides in length. In some cases, the guide polynucleotide is more than 90 nucleotides in length. In some cases, the guide polynucleotide is less than 245 nucleotides in length. In some embodiments, the 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 guide polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 to about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides in length.- 57 - #592931Attorney Docket No : 00010.036.1801

[0213] In some embodiments, sequence is determined by a BLASTP, CLUSTALW, MUSCLE, or MAFFT algorithm, or a CLUSTALW algorithm with the Smith-Waterman homology search algorithm parameters. In some embodiments, sequence is determined by the BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1. and using a conditional compositional score matrix adjustment.MG Systems

[0214] Described herein, in certain embodiments, are engineered nuclease systems comprising an endonuclease comprising and an engineered guide polynucleotide. In some embodiments, the engineered guide polynucleotide comprises a tracrRNA. In some embodiments, the engineered guide polynucleotide comprises a guide nucleic acid (e.g., gRNA). In a polynucleotide when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0215] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081,- 58 - #592931Attorney Docket No : 00010.036.18011082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity7to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504. 790-838. 888-903. 920-959. 1047-1081, 1082-1108, 1136-1165, 1209-1293. and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity' to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504. 790-838. 888-903. 920-959. 1047-1081, 1082-1108, 1136-1165. 1209-1293. and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity' to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NOs: 122-155, 194-215, 385-504, 790-838, 888-903, 920-959, 1047-1081, 1082-1108, 1136-1165, 1209-1293, and 1413-1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity7to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NOs: 122-155, 194-215. 385-504. 790-838. 888-903. 920-959, 1047-1081, 1082-1108. 1136-1165, 1209-1293, and 1413-1420.MG119-28 Targeting of albumin

[0216] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an - 59 - #592931Attorney Docket No : 00010.036.1801engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 365-368, 790-838. 1166, 1168. 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity’ to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 365-368, 790-838, 1166. 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity' to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 365-368, 790-838. 1166, 1168. 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%. 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172.- 60 - #592931Attorney Docket No : 00010.036.1801

[0217] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity’ to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170. and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO:- 61 - #592931Attorney Docket No : 00010.036.1801365-368, 790-838, 1166, 1168, 1170, and 1172. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%. 96%. 97%. 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 365-368, 790-838, 1166, 1168, 1170. and 1172. MG119-28 Targeting of A AVS1

[0218] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered - 62 - #592931Attorney Docket No : 00010.036.1801polynucleotide having at least about 98% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245. and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413.

[0219] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165. 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96%- 63 - #592931Attorney Docket No : 00010.036.1801identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165, 1212-1245, and 1413. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%. 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 920-959, 1082-1108, 1136-1165. 1212-1245, and 1413.MG119-28 Targeting ofAPOAl

[0220] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 888-903. In some embodiments, the - 64 - #592931Attorney Docket No : 00010.036.1801engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity7to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity7to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to any7one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%. 85%. 90%. 95%. 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 888-903.

[0221] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity7to any one of SEQ ID NOs: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity7to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 888-903.- 65 - #592931Attorney Docket No : 00010.036.1801In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity7to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 888-903. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 888-903.MG119-28 Targeting ofSODl

[0222] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an - 66 - #592931Attorney Docket No : 00010.036.1801endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity' to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity' to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity' to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%. 90%. 95%. 96%. 97%, 98%, 99% or 100% sequence identity’ to any one of SEQ ID NO: 1246-1293.

[0223] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to any one of SEQ ID NOs: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity7to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity’ to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity' to any one of - 67 - #592931Attorney Docket No : 00010.036.1801SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity7to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to any one of SEQ ID NO: 1246- 1293. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to any one of SEQ ID NO: 1246-1293. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 1246-1293.MG119-28 Targeting ofVCP

[0224] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence - 68 - #592931Attorney Docket No : 00010.036.1801having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity' to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1387.

[0225] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having - 69 - #592931Attorney Docket No : 00010.036.1801at least about 85% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity7to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity7to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1387.MG119-28 Targeting ofVEGFA

[0226] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at - 70 - #592931Attorney Docket No : 00010.036.1801least about 85% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity' to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity7to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity7to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity7to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity7to SEQ ID NO: 1414.

[0227] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity7to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease - 71 - #592931Attorney Docket No : 00010.036.1801system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1414. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1414.MG119-28 Targeting ofAtxn2

[0228] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity' to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1400 or 1402. In - 72 - #592931Attorney Docket No : 00010.036.1801some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%. 90%. 95%. 96%. 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 1400 or 1402.

[0229] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1400 or 1402. In - 73 - #592931Attorney Docket No : 00010.036.1801some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity7to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity7to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1400 or 1402. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 1400 or 1402.MG119-28 Targeting ofIFNG

[0230] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at - 74 - #592931Attorney Docket No : 00010.036.1801least about 85% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity' to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity7to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity7to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity7to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity7to SEQ ID NO: 1415.

[0231] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity7to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease - 75 - #592931Attorney Docket No : 00010.036.1801system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1415. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1415.MG 119-28 Targeting of CLIC4

[0232] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity' to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease - 76 - #592931Attorney Docket No : 00010.036.1801system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity' to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1416.

[0233] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity' to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity' to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ - 77 - #592931Attorney Docket No : 00010.036.1801ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1416. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%. 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1416.MG119-28 Targeting ofFGF18

[0234] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ - 78 - #592931Attorney Docket No : 00010.036.1801ID NO: 28 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity^ to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity' to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity' to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%. 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity' to SEQ ID NO: 1417.

[0235] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity' to SEQ ID NO:- 79 - #592931Attorney Docket No : 00010.036.18011417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity' to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1417. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1417.MG119-28 Targeting ofNLRC4

[0236] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO:- 80 - #592931Attorney Docket No : 00010.036.18011418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity' to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity' to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1418.

[0237] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease - 81 - #592931Attorney Docket No : 00010.036.1801comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity' to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1418. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1418.MG119-28 Targeting ofNUDT16

[0238] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity' to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease - 82 - #592931Attorney Docket No : 00010.036.1801comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 95% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity' to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 1419.

[0239] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity' to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered - 83 - #592931Attorney Docket No : 00010.036.1801polynucleotide having at least about 95% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity7to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1419. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1419.MG119-28 Targeting ofEMC6

[0240] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 70% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 75% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 85% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 90% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 28 and an engineered - 84 - #592931Attorney Docket No : 00010.036.1801polynucleotide having at least about 95% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 97% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 28 and an engineered polynucleotide having at least about 99% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 28 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 28 and an engineered polynucleotide having at least 70%. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity7to SEQ ID NO: 1420.

[0241] In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 70% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 70% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 75% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 75% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 80% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 80% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 85% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 85% identity7to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 90% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 90% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 95% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 95% identity7to SEQ ID NO: 1420. In some embodiments, the - 85 - #592931Attorney Docket No : 00010.036.1801engineered nuclease system comprises an endonuclease comprising sequence having at least about 96% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 96% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 97% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 97% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 98% identity7to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 98% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising sequence having at least about 99% identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least about 99% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to SEQ ID NO: 1382 and an engineered polynucleotide comprising 100% identity to SEQ ID NO: 1420. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1382 and an engineered polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1420.

[0242] Further described herein, in some embodiments, are engineered nuclease systems comprising an endonuclease provided herein and a DNA methyltransferase. In some embodiments, the DNA methyltransferase binds non-covalently to the endonuclease. In some embodiments, the DNA methyltransferase is fused to the endonuclease in a single polypeptide. In some embodiments, the DNA methyltransferase comprises Dmnt3 A or Dnmt3L. In some embodiments, the engineered nuclease system further comprises a KRAB domain. In some embodiments, the KRAB domain binds non-covalently to the endonuclease or the DNA methyltransferase. In some embodiments, the KRAB domain is covalently linked to the endonuclease or the DNA methyltransferase. In some embodiments, the KRAB domain is fused to the endonuclease or the DNA methyltransferase in a single polypeptide.Cells

[0243] Described herein, in certain embodiments, is a cell comprising the class 2, type V nuclease systems described herein.

[0244] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryo kidney (HEK), mouse myeloma (NS0), or human retinal cells), an - 86 - #592931Attorney Docket No : 00010.036.1801immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, aHuh7 cell, aHepG2 cell, aK562 cell, aN2a cell, or a SY5Y cell), an insect cell (e.g., aSpodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a S2 cell, or aHeliothis virescens cell), a yeast cell (e.g., aSaccharomyces cerevlsiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharom yces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokary otic cell (e.g., a E. coli cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell). In some embodiments, the cell is a eukary otic 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.

[0245] In some embodiments, the cell is an A549, HEK-293, HEK-293T. BHK, CHO. HeLa, MRC5. Sf9, Cos-1, Cos-7, Vero, BSC 1. BSC 40. BMT 10. WI38, HeLa, Saos, C2C12. L cell. HT1080, HepG2, Huh7, K562, a primary' cell, or derivative thereof. In some embodiments, the primary' cell is a T cell. In some embodiments, the primary' cell is a hematopoietic stem cell (HSC).Delivery and Vectors

[0246] Disclosed herein, in some embodiments, are nucleic acid sequences encoding a MG119 system comprising a class 2, type V effector a gRNA, or a gene editing system disclosed herein.

[0247] In some embodiments, the nucleic acid encoding the MG119 system is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid encoding the MG119 system is an RNA, for example a mRNA.

[0248] In some embodiments, the nucleic acid encoding the MG119 system is delivered by a nucleic acid-based vector. In some embodiments, the nucleic acid-based vector is a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), Pl -derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus. In some embodiments, the nucleic acidbased vector is selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG. pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4, pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-Nl vector, pEFla-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal,pSF-OXB20-Fluc, pSF- - 87 - #592931Attorney Docket No : 00010.036.1801OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0249] In some embodiments, the nucleic acid-based vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, pl 9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof. In some embodiments the promoter is a U6 promoter. In some embodiments, the promoter is a CAG promoter.

[0250] In some embodiments, the nucleic acid-based vector is a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.

[0251] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8. AAV-Myo, AAV-NP40, AAV-NP59. AAV-NP22, AAV-NP66, AAV-HSC16. or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0252] 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 AAV 5 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 - 88 - #592931Attorney Docket No : 00010.036.1801thereof. In some embodiments, the virus is AAV 10 or a derivative thereof. In some embodiments, the virus is AAV 11 or a derivative thereof. In some embodiments, the virus is AAV 12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV 14 or a derivative thereof. In some embodiments, the virus is AAV 15 or a derivative thereof. In some embodiments, the virus is AAV 16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rhlO or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-l or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof. In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the vims 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 vims is AAV-NP40 or a derivative thereof. In some embodiments, the vims is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the vims is- 89 - #592931Attorney Docket No : 00010.036.1801AAV -NP66 or a derivative thereof. In some embodiments, the virus is AAV -HSC 16 or a derivative thereof.

[0253] 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.

[0254] In some embodiments, the nucleic acid encoding the class 2, type V effector or the genome editing system is delivered by anon-nucleic acid-based delivery system (e.g., anon-viral delivery system). In some embodiments, the non-viral delivery system is a liposome. In some embodiments, the nucleic acid is associated with a lipid. The nucleic acid associated with a lipid, in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined wi th a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP).

[0255] In some embodiments, the class 2, t pe V effector or the genome editing system is introduced into the cell in any suitable way, either stably or transiently. In some embodiments, the class 2, type V effector or the genome editing system is transfected into the cell. In some embodiments, the cell is transduced or transfected with a nucleic acid construct that encodes the class 2, type V effector or the genome editing system. For example, a cell is transduced (e.g., with a virus encoding the class 2, type V effector or the genome editing system), or transfected (e.g., with a plasmid encoding the class 2, type V effector or the genome editing system) with a nucleic acid that encodes the class 2, type V effector or the genome editing system, or the translated the class 2. type V effector or the genome editing system. In some embodiments, the transduction is a stable or transient transduction. In some embodiments, cells expressing the class 2, type V effector or the genome editing system or containing the class 2, type V effector or the genome editing system are transduced or transfected with one or more gRNA molecules, for example, when the class 2, type V effector or the genome editing system comprises a CRISPR nuclease. In some embodiments, a plasmid expressing the class 2, type V effector or the genome editing system is introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction (for example lenti virus or AAV) or other - 90 - #592931Attorney Docket No : 00010.036.1801methods known to those of skill in the art. In some embodiments, the gene editing system is introduced into the cell as one or more polypeptides. In some embodiments, delivery is achieved through the use of RNP complexes. Delivery methods to cells for polypeptides and / or RNPs are known in the art, for example by electroporation or by cell squeezing.

[0256] Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electroporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos.5,049,386; 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of WO 91 / 17424 and WO 91 / 16024. In some embodiments, the delivery' is to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g.. in vivo administration). In some embodiments, the nucleic acid is comprised in a liposome or a nanoparticle that specifically targets a host cell.

[0257] Additional methods for the delivery' of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003 / 0087817.

[0258] In some embodiments, the present disclosure provides a cell comprising a vector or a nucleic acid described herein. In some embodiments, the cell expresses a gene editing system or parts thereof. In some embodiments, the cell is a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.Methods of Use

[0259] Gene editing systems of the present disclosure may be used for various applications, such as, for example, nucleic acid editing (e.g., gene editing) or binding to a nucleic acid molecule (e.g., sequence-specific binding). Such systems may be used, for example, for remediating (e.g., removing or replacing) a genetically inherited mutation that may cause a disease in a subject; inactivating a gene in order to ascertain its function in a cell; as a diagnostic tool to detect diseasecausing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or an amplified DNA sequence encoding a disease-causing mutation); as deactivated enzy mes in combination with a probe to target and detect a specific nucleotide sequence (e.g., sequence encoding antibiotic resistance int bacteria); to render viruses inactive or incapable of infecting host cells by targeting viral genomes; to add genes or amend metabolic pathyvays to engineer organisms to produce valuable small molecules, macromolecules, or secondary' metabolites; to establish a gene drive - 91 - #592931Attorney Docket No : 00010.036.1801element for evolutionary selection, and / or to detect cell perturbations by foreign small molecules and nucleotides as a biosensor.

[0260] Described herein, in certain embodiments, are methods of modifying a target nucleic acid site comprising providing a class 2, type V effector or the genome editing system disclosed herein. In some embodiments, modifying the target nucleic acid site comprises binding, nicking, cleaving, marking, modifying, or transposing the target nucleic acid site. In some cases, the endonuclease induces a single-stranded break or a double-stranded break at or proximal to the target nucleic acid site. In some cases, the endonuclease induces a staggered single-stranded break within or 3' to the target nucleic acid site.

[0261] In some embodiments, the target nucleic acid site is double-stranded. In some embodiments, the target nucleic acid site is single-stranded. In some cases, the target nucleic acid site comprises deoxyribonucleic acid (DNA). In some cases, the target nucleic acid site is doublestranded DNA. In some cases, the target nucleic acid site comprises ribonucleic acid (RNA). In some cases, the target nucleic acid site comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some cases, the target nucleic acid site is in vitro. In some cases, the target nucleic acid site is within a cell. In some cases, the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell. In some embodiments, the cell is genome edited ex vivo. In some embodiments, the cell is genome edited in vivo.

[0262] In some embodiments, the methods are used to introduce a modification in the genome of a cell. In some embodiments, the modification is an insertion, deletion, or mutation. In some embodiments, the methods are used to introduce site-directed insertions, deletions, and / or mutations in the genome of a cell (for example an insertion and a mutation). In some embodiments, the methods are used in combination with a nucleic acid template to facilitate site-directed insertions into the genome of a cell.

[0263] In some embodiments, the methods are used for binding, cleaving, marking, or modifying a double-stranded deoxyribonucleic acid polynucleotide. In some embodiments, the methods comprise contacting the double-stranded deoxyribonucleic acid polynucleotide with an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a class 2 Cas endonuclease. In some cases, the endonuclease is a class 2, type V Cas endonuclease. In some cases, the endonuclease is a class 2, type V, subtype Cas endonuclease. In some cases, the endonuclease is in complex with an engineered guide RNA. In some cases, the engineered guide RNA is configured to bind to the endonuclease. In some cases, the engineered guide RNA is configured to bind to the double-stranded deoxyribonucleic acid polynucleotide. In - 92 - #592931Attorney Docket No : 00010.036.1801some cases, the engineered guide RNA is configured to bind to the endonuclease and to the doublestranded deoxyribonucleic acid polynucleotide. In some cases, the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM).

[0264] Described herein, in certain embodiments, are methods of modifying TRAC comprising contacting TRAC using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence has any one of SEQ ID NOs: 505-536. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 505-536.

[0265] Described herein, in certain embodiments, are methods of modifying APOA1 comprising contacting APOA1 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 888-903. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 888-903 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 888-903. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 888-903 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identify to any one of SEQ ID NOs: 888-903. In some embodiments, the target nucleic acid sequence has any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identify7to any one of SEQ ID NOs: 537-653, 703-721, 904-919, and 1030-1036.

[0266] Described herein, in certain embodiments, are methods of modify ing AAVS1 comprising contacting AAVS1 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identify to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least - 93 - #592931Attorney Docket No : 00010.036.180180% sequence identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 920-959. 1082-1108, 1136-1165, and 1212-1245, and 1413, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 920-959, 1082-1108, 1136-1165, and 1212-1245, and 1413. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405.

[0267] In some embodiments, the target nucleic acid sequence has any one of SEQ ID NOs: 722-789, 960-999, 1037-1046. 1109-1135, and 1405. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity' to any one of SEQ ID NOs: 722-789, 960-999, 1037-1046, 1109-1135, and 1405.

[0268] Described herein, in certain embodiments, are methods of modifying VEGFA comprising contacting VEGFA using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1414. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1414 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1414. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1414, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1414. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1406. In - 94 - #592931Attorney Docket No : 00010.036.1801some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1406.

[0269] Described herein, in certain embodiments, are methods of modifying VEGFA comprising contacting VEGFA using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identify to any one of SEQ ID NOs: 28, 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1414. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identify’ to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1414 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identify to SEQ ID NO: 1414. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1414, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identify’ to SEQ ID NO: 1414. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1406. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identify to SEQ ID NO: 1406.

[0270] Described herein, in certain embodiments, are methods of modifying IFNG comprising contacting IFNG using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identify to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1415. In some embodiments, the endonuclease has at least 80% sequence identify to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identify to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1407. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identify to SEQ ID NO: 1407.

[0271] Described herein, in certain embodiments, are methods of modifying IFNG comprising contacting IFNG using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identify to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1415. In some embodiments, the endonuclease has at least 80% sequence identify to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%,- 95 - #592931Attorney Docket No : 00010.036.180198%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1415 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1415. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1415, or a sequence having at least 90%. 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1415. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1407. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1407.

[0272] Described herein, in certain embodiments, are methods of modifying CLIC4 comprising contacting CLIC4 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ IDNOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1416. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1408. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1408.

[0273] Described herein, in certain embodiments, are methods of modifying CLIC4 comprising contacting CLIC4 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1416. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1416 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1416. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1416, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1416. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1408. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1408.- 96 - #592931Attorney Docket No : 00010.036.1801

[0274] Described herein, in certain embodiments, are methods of modifying FGF18 comprising contacting FGF18 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ IDNOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1417. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1409. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1409.

[0275] Described herein, in certain embodiments, are methods of modifying FGF18 comprising contacting FGF18 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ IDNOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1417. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%. or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1417 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1417. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1417, or a sequence having at least 90%. 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1417. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1409. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1409.

[0276] Described herein, in certain embodiments, are methods of modifying NLRC4 comprising contacting NLRC4 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1418. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity7to SEQ ID NO: 28 or 1382. In some embodiments,- 97 - #592931Attorney Docket No : 00010.036.1801the target nucleic acid sequence has SEQ ID NO: 1410. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1410.

[0277] Described herein, in certain embodiments, are methods of modifying NLRC4 comprising contacting NLRC4 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1418. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1418 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1418. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary’ to a sequence comprising SEQ ID NO: 1418, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1418. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1410. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1410.

[0278] Described herein, in certain embodiments, are methods of modifying NUDT16 comprising contacting NUDT16 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1419. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1411. In some embodiments, the target nucleic acid sequence has at least 90%. 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1411.

[0279] Described herein, in certain embodiments, are methods of modifying NUDT16 comprising contacting NUDT16 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity’ to SEQ ID NO: 1419. In some embodiments, the - 98 - #592931Attorney Docket No : 00010.036.1801endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1419 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1419. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1419, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1419. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1411. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity’ to SEQ ID NO: 1411.

[0280] Described herein, in certain embodiments, are methods of modifying EMC 6 comprising contacting EMC6 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ IDNOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity’ to SEQ ID NO: 1420. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%. 98%. or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1412. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1412.

[0281] Described herein, in certain embodiments, are methods of modifying EMC6 comprising contacting EMC6 using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1420. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1420 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1420. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1420, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1420. In some embodiments, the target nucleic acid- 99 - #592931Attorney Docket No : 00010.036.1801sequence has SEQ ID NO: 1412. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1412.

[0282] Described herein, in certain embodiments, are methods of modifying albumin comprising contacting albumin using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity to any one of SEQ ID NOs: 1-121. 182-193. 216-364. and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, and 1170-1172. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the engineered guide polynucleotide is encoded by any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, and 1170-1172 or a sequence having at least 90%. 95%. 97%. 98%, or 99% sequence identity to any one of SEQ ID NOs: 790-838, 365-368. 1166, 1168. and 1170-1172. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168, and 1170-1172 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 790-838, 365-368, 1166, 1168. and 1170-1172. In some embodiments, the target nucleic acid sequence has any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 654-702, 839-887, 1000-1029, 1167, 1169, 1171, 1173, 1206, and 1207.

[0283] Described herein, in certain embodiments, are methods of modifying VCP comprising contacting VCP using an engineered nuclease system comprising: a) an endonuclease having at least 80% sequence identity7to any one of SEQ ID NOs: 1-121, 182-193, 216-364, and 1294-1382; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1387. In some embodiments, the endonuclease has at least 80% sequence identity to SEQ ID NO: 28 or 1382. In some embodiments, the endonuclease has at least 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 28 or 1382. In some embodiments, the target nucleic acid sequence has SEQ ID NO: 1385. 1391, or 1392. In some embodiments, the target nucleic acid sequence has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1385, 1391, or 1392.- 100 - #592931Attorney Docket No : 00010.036.1801

[0284] Described herein, in certain embodiments, are methods of modifying a VCP gene comprising contacting the VCP gene using an engineered nuclease system comprising: a) an endonuclease having at least 70% sequence identity to SEQ ID NO: 28; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1387. In some embodiments, the engineered guide polynucleotide is encoded by SEQ ID NO: 1387 or a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1387. In some embodiments, the engineered nuclease system comprises an endonuclease encoded by a sequence having 70%, 75%, 80%. 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2169. In some embodiments, the engineered guide polynucleotide is configured to hybridize to a sequence or target a sequence complementary to a sequence comprising SEQ ID NO: 1387 or a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1387. In some embodiments, the target nucleic acid sequence comprises SEQ ID NOs: 1385, 1391, or 1392. In some embodiments, the target nucleic acid sequence has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1385, 1391, or 1392.

[0285] Further described herein, in certain embodiments, are methods of manufacturing an engineered nuclease system described herein or components thereof In some embodiments, the method comprises cultivating a host cell with the engineered nuclease system described herein or components thereof.

[0286] In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve. Bifidobacterium infantis, Bifidobacterium adolescentis. Lactobacillus acidophilus. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus fermentum, Laclococcus lactis, Streptococcus lhermophilus, Laclococcus lactis, Lactococcus diacetylactis. Lactococcus cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii, or Escherichia coli. In some embodiments, the host cell is an E. coli cell. In some embodiments, the E. coli cell is a XDE3 lysogen or a BL21(DE3) strain. In some embodiments, the E. coli cell has an ompT Ion genotype.

[0287] In some embodiments, the host cell is an E. coli cell. In some embodiments, the E. coli cell is a DE3 lysogen or the E. coli cell is a BL21(DE3) strain. In some embodiments, the E. coli cell has an ompT Ion genotype.- 101 - #592931Attorney Docket No : 00010.036.1801

[0288] In some embodiments, the open reading frame is operably linked to a promoter sequence. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the promoter is selected from the group consisting of CMV. CBA, EFla, CAG, PGK, TRE, U6, UAS, T7. Sp6, lac, araBad. trp, Ptac. p5. p!9, p40, Synapsin, CaMKII, GRK1, and derivatives thereof.

[0289] In some embodiments, the open reading frame is operably linked to a T7 promoter sequence, a T7-lac promoter sequence, a lac promoter sequence, a tac promoter sequence, a trc promoter sequence, a ParaBAD promoter sequence, a PrhaBAD promoter sequence, a T5 promoter sequence, a cspA promoter sequence, an araPBAD promoter, a strong leftward promoter from phage lambda (pL promoter), or any combination thereof.

[0290] In some embodiments, the open reading frame comprises a sequence encoding an affinity tag linked in-frame to a sequence encoding the engineered nuclease system described herein or components thereof. In some embodiments, the affinity’ tag is an immobilized metal affinity chromatography (IMAC) tag. In some embodiments, the IMAC tag is a polyhistidine tag. In some embodiments, the affinity tag is a myc tag, a human influenza hemagglutinin (HA) tag, a maltose binding protein (MBP) tag, a glutathione S-transferase (GST) tag, a streptavidin tag, a FLAG tag, or any combination thereof. In some embodiments, the affinity tag is linked in-frame to the sequence encoding the engineered nuclease system described herein or components thereof via a linker sequence encoding a protease cleavage site. In some embodiments, the protease cleavage site is a tobacco etch virus (TEV) protease cleavage site, a PreScission® protease cleavage site, a Thrombin cleavage site, a Factor Xa cleavage site, an enterokinase cleavage site, or any combination thereof.

[0291] In some embodiments, the open reading frame is codon-optimized for expression in the host cell. In some embodiments, the open reading frame is provided on a vector. In some embodiments, the open reading frame is integrated into a genome of the host cell.

[0292] In some embodiments, the present disclosure provides a culture comprising a host cell described herein in compatible liquid medium.

[0293] In some embodiments, the present disclosure provides a method of producing an engineered nuclease system described herein or components thereof, comprising cultivating a host cell described herein in compatible growth medium. In some embodiments, the method further compris...

Claims

Attorney Docket No : 00010.036.1801CLAIMSWHAT IS CLAIMED IS:

1. An engineered nuclease system, comprising:a) an endonuclease comprising one or more amino acid modifications at a position selected from the group consisting of: K93, E202, M204, E236, E255, and N329 as compared to SEQ ID NO: 28; andb) an engineered guide polynucleotide that forms a complex with the endonuclease and hybridizes to a target nucleic acid sequence.

2. The engineered nuclease system of claim 1 or 2, wherein the endonuclease comprises one or more amino acid modifications selected from the group consisting of: K93R, E202K, M204K, E236K, E255K, and N329E as compared to SEQ ID NO: 28.

3. The engineered nuclease system of claim 1 or 2, wherein the endonuclease is 100% identical to SEQ ID NO: 1382.

4. An engineered nuclease system, comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and to hybridize to a target nucleic acid sequence.

5. The engineered nuclease system of claim 4, wherein the endonuclease has at least 80% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381.

6. The engineered nuclease system of claim 4, wherein the endonuclease has at least 90% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381.

7. The engineered nuclease system of claim 4, wherein the endonuclease has 100% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381.

8. The engineered nuclease system of any one of claims 1-7, wherein the engineered guide polynucleotide comprises a crRNA and a tracrRNA.

9. The engineered nuclease system of any one of claims 1-7, wherein the engineered guide polynucleotide has at least 90% sequence identity to any one of SEQ ID NOs: 201, 424-449, 790-838, 888-903, 920-959, 1063-1078. 1079-1108, 1136-1165, 1209-1211. 1212-1293, 1413-1420, 122-155, 194-200, 202-215, 385-423, 450-504, 1047-1062, and 1387.

10. The engineered nuclease system of claim 9, wherein the engineered guide polynucleotide has 100% sequence identity to any one of SEQ ID NOs: 201, 424-449, 790-838, 888-903, 920-959, 1063-1078, 1079-1108, 1136-1165, 1209-1211, 1212-1293, 1413-1420, 122-155, 194-200, 202-215, 385-423, 450-504, 1047-1062, and 1387.Attorney Docket No : 00010.036.1801 11. The engineered nuclease system of any one of claims 1-10, wherein the engineered guide polynucleotide is a single guide nucleic acid.

12. The engineered nuclease system of any one of claims 1-10. wherein the engineered guide polynucleotide is a dual guide nucleic acid.

13. The engineered nuclease system of any one of claims 1-12, wherein the engineered guide polynucleotide is RNA.

14. The engineered nuclease system of any one of claims 1-13, wherein the endonuclease binds non-covalently to the engineered guide polynucleotide.

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

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

17. The engineered nuclease system of any one of claims 1-16, further comprising a DNA methyltransferase.

18. The engineered nuclease system of claim 17, wherein the DNA methyltransferase binds non-covalently to the endonuclease.

19. The engineered nuclease system of claim 17, wherein the DNA methyltransferase is fused to the endonuclease in a single polypeptide.

20. The engineered nuclease system of any one of claims 17-19, wherein the DNA methyltransferase comprises Dmnt3A or Dnmt3L.

21. A method of modifying a target nucleic acid sequence comprising contacting the target nucleic acid sequence using the engineered nuclease system of any one of claims 1-20.

22. The method of claim 21, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence.

23. The method of any one of claims 21-22, wherein the target nucleic acid sequence comprises genomic DNA. viral DNA, viral RNA, or bacterial DNA.

24. The method of any one of claims 21-23, wherein the modification is in vitro.

25. The method of any one of claims 21-23, wherein the modification is in vivo.

26. The method of any one of claims 21-23, wherein the modification is ex vivo.

27. A method of modifying a target nucleic acid sequence in a mammalian cell comprising contacting the mammalian cell using the engineered nuclease system of any one of claims 1-20.

28. The method of claim 27, further comprising selecting cells comprising the modification.

29. A method of modifying adeno-associated virus integration site 1 (AAVS1) comprising contacting AAVS1 using an engineered nuclease system comprising:Attorney Docket No : 00010.036.1801 a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 1413, 1212-1245, 920-959, 1082-1108, and 1136-1165.

30. A method of modifying superoxide dismutase 1 (SOD1) comprising contacting SOD1 using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity’ to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to any one of SEQ ID NOs: 1246-1293.

31. A method of modifying a valosin containing protein (V CP) gene comprising contacting the VCP gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NO: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1387.

32. A method of modifying a vascular endothelial growth factor A (VEGFA) gene comprising contacting the VEGFA gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1414.

33. A method of modifying an interferon gamma (IFNG) gene comprising contacting the IFNG gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1415.Attorney Docket No : 00010.036.1801 34. A method of modifying a chloride intracellular channel 4 (CLIC4) gene comprising contacting the CLIC4 gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1416.

35. A method of modifying a fibroblast growth factor 18 (FGF18) gene comprising contacting the FGF18 gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1417.

36. A method of modifying a NLR family CARD domain-containing protein 4 (NLRC4) gene comprising contacting the NLRC4 gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identity to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identity to SEQ ID NO: 1418.

37. A method of modifying a nudix hydrolase 16 (NUDT16) gene comprising contacting the NUDT16 gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identify to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1419.

38. A method of modifying an ER membrane protein complex subunit 6 (EMC6) gene comprising contacting the EMC6 gene using an engineered nuclease system comprising:a) an endonuclease having at least 70% sequence identify to any one of SEQ ID NOs: 1382 and 1294-1381; andb) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide having at least 80% sequence identify to SEQ ID NO: 1420.Attorney Docket No : 00010.036.180139. A nucleic acid encoding the engineered nuclease system of any one of claims 1-20.

40. A cell comprising the engineered nuclease system of any one of claims 1-20.

41. The cell of claim 40, wherein the cell is a eukaryotic cell.

42. The cell of claim 40, wherein the cell is a mammalian cell.

43. The cell of claim 40, wherein the cell is an immortalized cell.

44. The cell of claim 40, wherein the cell is an insect cell.

45. The cell of claim 40, wherein the cell is a yeast cell.

46. The cell of claim 40, wherein the cell is a plant cell.

47. The cell of claim 40, wherein the cell is a fungal cell.

48. The cell of claim 40, wherein the cell is a prokaryotic cell.

49. The cell of claim 40, wherein the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, S®. Cos-1, Cos-7, Vero, BSC 1. BSC 40, BMT 10, WI38, HeLa. Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or a derivative thereof.

50. The cell of claim 40, wherein the cell is an engineered cell.

51. The cell of claim 40, wherein the cell is a stable cell.

52. The cell of claim 40, wherein the cell is a primary cell.

53. The cell of claim 40. wherein the primary cell is a T cell.

54. The cell of claim 40, wherein the primary cell is a hematopoietic stem cell (HSC).

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