Class II and V CRISPR systems

Abstract

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

Description

[Technical Field]

[0001] cross reference

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 369,920, filed July 29, 2022, U.S. Provisional Patent Application No. 63 / 386,298, filed December 6, 2022, and U.S. Provisional Patent Application No. 63 / 484,168, filed February 9, 2023, each of which is incorporated herein by reference in its entirety. [Background technology]

[0003] Cas enzymes, along with their associated clustered regularly interspaced short palindromic repeats (CRISPR)-guided ribonucleic acid (RNA), appear to be widespread components of prokaryotic immune systems (approximately 45% of bacteria and 84% of archaea), helping to protect such microorganisms against non-self nucleic acids, such as infectious viruses and plasmids, through CRISPR-RNA-guided nucleic acid cleavage. While deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse and contain a wide variety of nucleic acid-interacting domains. While CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capabilities of CRISPR / Cas complexes have only been recognized relatively recently, leading to the use of recombinant CRISPR / Cas systems in diverse DNA manipulation and gene editing applications.

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on July 24, 2023, is named MTG-016WO_SL.xml and is 14,188,189 bytes in size. Summary of the Invention

[0005] In certain embodiments, described herein are engineered nuclease systems comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; 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 engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. 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 engineered endonuclease is non-covalently linked 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 guide polynucleotide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 3612. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to SEQ ID NO: 3612. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of SEQ ID NOs: 3870-3872. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising YYn.

[0006] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6274-6281; 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 engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6274-6281. 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 engineered endonuclease is non-covalently bound 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 guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of the following sequences: tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC.

[0007] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6931-6952.

[0008] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6069-6077.

[0009] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6080-6081.

[0010] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6178-6273.

[0011] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, the endonuclease comprises at least one of the following mutations when the sequence of the endonuclease is optimally aligned with SEQ ID NO: 215. In some embodiments, the engineered nuclease system further comprises a single-stranded or double-stranded DNA repair template comprising, from 5' to 3', a first homologous 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 homologous arm comprising a sequence of at least 20 nucleotides 3' to the target sequence. In some embodiments, the first or second homologous arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. In some embodiments, the first and second homologous arms are homologous to a genomic sequence of a prokaryote, a bacterium, a fungus, or a eukaryote.

[0012] In certain embodiments, described herein are methods for modifying a target nucleic acid sequence, comprising contacting the target nucleic acid sequence using an engineered nuclease system 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 is within CD38, TIGIT, AAVS1, B2M, CD2, CD5, hRosa26, TRAC, TRBC1, TRBC2, FAS, PD-1, HPRT, HAO-1, APO-A1, ANGPTL3, GPR146, or VCP. 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.

[0013] In certain embodiments, described herein are methods for modifying a target nucleic acid sequence in a mammalian cell, comprising contacting the mammalian cell with an engineered nuclease system described herein. In some embodiments, the method further comprises selecting the cell containing the modification.

[0014] In certain embodiments, described herein are methods of modifying the HAO-1 gene, comprising contacting the HAO-1 gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6931-6952.

[0015] In certain embodiments, described herein are methods of modifying the human GPR146 gene, comprising contacting the human GPR146 gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6069-6077.

[0016] In certain embodiments, described herein are methods of modifying a mouse GPR146 gene, comprising contacting the mouse GPR146 gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6080-6081.

[0017] In certain embodiments, described herein are methods of modifying the ANGPTL3 gene, comprising contacting the ANGPTL3 gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6178-6273.

[0018] In certain embodiments, described herein are methods of modifying a VCP gene, comprising contacting the VCP gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6557-6562.

[0019] In certain embodiments, described herein 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 cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cell is an engineered cell. In some embodiments, the cell is a stable cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a hematopoietic cell.

[0020] In certain embodiments, described herein are lipid nanoparticles comprising (a) an engineered nuclease system described herein, (b) a cationic lipid, (c) a sterol, (d) a neutral lipid, and (e) a PEG-modified lipid. In some embodiments, the cationic lipid comprises C12-200, the sterol comprises cholesterol, the neutral lipid comprises DOPE, or the PEG-modified lipid comprises DMG-PEG2000. In some embodiments, the cationic lipid comprises 98N12-5 (TETA5-LAP), DLin DMA, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, or C12-200.

[0021] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. [Brief explanation of the drawings]

[0022] The novel features of the present 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.

[0023] [Figure 1] Depicts the results of gene editing at the DNA level of human GPR146 in Hep3B cells. [Figure 2] Depicts the results of gene editing at the DNA level of mouse GPR146 in Hepa1-6 cells. [Figure 3] Depicts the results of gene editing at the DNA level of human ANGPTL3 in Hep3B cells. [Figure 4]Depicts the results of gene editing at the DNA level of human GPR146 in primary human hepatocytes. [Figure 5] Depicts the results of gene editing at the DNA level of mouse GPR146 in primary mouse hepatocytes. [Figure 6A] The predicted fold of the spacer-less single guide RNA (sgRNA) sequence is depicted. The tracrRNA and repeat sequence are looped at a GAAA tetraloop. A repeat-antirepeat fold is located on the 3' end of each structure (circled at the far right). For Figure 6A, these two candidate tracrRNA sequences were obtained from in silico analysis of the intergenic region, suggesting that they potentially encode tracrRNA. [Figure 6B] The predicted fold of a single guide RNA (sgRNA) sequence without a spacer is depicted. The tracrRNA and repeat sequence are looped at a GAAA tetraloop. The repeat-antirepeat fold is located on the 3' end of each structure (circle at the far right). For Figures 6B and 6C, the tracrRNA was predicted using a covariance model built from previously active sgRNAs. The sgRNA number (sg#) is indicated below the nuclease number. [Figure 6C] The predicted fold of a single guide RNA (sgRNA) sequence without a spacer is depicted. The tracrRNA and repeat sequence are looped at a GAAA tetraloop. The repeat-antirepeat fold is located on the 3' end of each structure (circle at the far right). For Figures 6B and 6C, the tracrRNA was predicted using a covariance model built from previously active sgRNAs. The sgRNA number (sg#) is indicated below the nuclease number. [Figure 7A]Figure 7 depicts in vitro cleavage assay amplification products. Figure 7A depicts a 2% agarose gel with a low molecular weight DNA ladder in the leftmost lane. Figure 7B depicts a digital gel. The resulting amplicon products are 188 bp with U67 spacer-bearing guides or 205 bp with U40 spacer-bearing guides. The specific sgRNA (sg#) and spacer used for each nuclease are indicated above the lane. [Figure 7B] Figure 7B depicts the in vitro cleavage assay amplification products. Figure 7B depicts the digital gel. The resulting amplicon products are 188 bp with U67 spacer-bearing guides or 205 bp with U40 spacer-bearing guides. The specific sgRNA (sg#) and spacer used for each nuclease are indicated above the lane. [Figure 8A] Depicts sequence logos of protospacer adjacent motif (PAM) sequences obtained from NGS sequencing of amplified cleavage sites on the template strand (Figure 8A) and non-template strand (Figure 8B). The specific sgRNA (sg#) and spacer are indicated next to the nuclease number. [Figure 8B] Depicts sequence logos of protospacer adjacent motif (PAM) sequences obtained from NGS sequencing of amplified cleavage sites on the template strand (Figure 8A) and non-template strand (Figure 8B). The specific sgRNA (sg#) and spacer are indicated next to the nuclease number. [Figure 9A] Histograms depict the number of DNA reads mapping to the amplified cleavage sites of each MG91 nuclease on the template strand (Figure 9A) and non-template strand (Figure 9B). The specific sgRNA (sg#) and spacer are indicated next to the nuclease number. [Figure 9B] Histograms depict the number of DNA reads mapping to the amplified cleavage sites of each MG91 nuclease on the template strand (Figure 9A) and non-template strand (Figure 9B). The specific sgRNA (sg#) and spacer are indicated next to the nuclease number. [Figure 10]Depicts the results of gene editing at the DNA level of human VCP in K562 cells. [Figure 11A] Figure 11 depicts an example of the purification of MG91-666, as well as activity analysis of MG91-666 and MG91-155. Figure 11A depicts an exemplary SDS-PAGE gel monitoring steps in the MG91-666 protein expression induction and purification process. The expected protein MW was approximately 106 kDa. [Figure 11B] Figure 11B depicts an example of purification of MG91-666, and activity analysis of MG91-666 and MG91-155. Figure 11B depicts a chromatogram of MBP fusion-enriched protein run on an anion exchange chromatography column. Peak fractions were collected and concentrated (shaded box). [Figure 11C] Figure 11C depicts an example of purification of MG91-666 and activity analysis of MG91-666 and MG91-155. Figure 11C depicts a chromatogram of the MBP fusion-enriched protein after anion exchange, cleaved to remove MBP, and run on a 200 Increase 10 / 300 G size-exclusion chromatography column. Peak fractions were collected and concentrated (shaded box). [Figure 11D] Figure 11D depicts an example of purification of MG91-666, as well as activity analysis of MG91-666 and MG91-155. Figure 11C depicts protein activity assessed in an in vitro cleavage reaction using 521 bp linear DNA as a substrate. Lanes: (1) ladder, (2) substrate alone, (3) substrate + 20-fold molar excess RNP. [Figure 11E] Figure 11E depicts an example of purification of MG91-666, as well as activity analysis of MG91-666 and MG91-155. Figure 11E depicts protein activity assessed in an in vitro cleavage reaction using 2,281-bp plasmid DNA as a substrate. Lanes: (1) ladder, (2) APO (no guide) protein, (3) substrate + 20-fold molar excess RNP. [Figure 12A]Figure 12 depicts an in vitro cleavage assay using the MG29 ancestral nuclease. The novel type V effectors MG29-229, MG29-230, and MG29-231 were assayed for cleavage activity via a PAM enrichment protocol. Figure 12A depicts the effectors expressed via an in vitro transcription / translation (IVTT) reaction in the presence of crRNA from the active nuclease MG29-1 and added to a PAM library (dsDNA target). Cleavage products were amplified via ligation to the cleavage site and subsequent PCR amplification. [Figure 12B] Figure 12 depicts an in vitro cleavage assay using the MG29 ancestral nuclease. The novel type V effectors MG29-229, MG29-230, and MG29-231 were assayed for cleavage activity via a PAM enrichment protocol. Figure 12B depicts a sequence logo of the preferred PAM sequence and cleavage location relative to the PAM sequence generated from NGS sequencing of the bands identified in Figure 12A. MG29-229 was weakly active, with a signal too low to confidently determine the PAM via NGS. [Figure 13] 1 depicts the relative effect of a single 2'OMe at each position in the 20 nt spacer of the MG29-1 guide. [Figure 14] 1 depicts the analysis of gene editing results (indels%) at the DNA level of hHAO1-4b in primary human hepatocytes. [Figure 15] 1 depicts the analysis of gene editing results (indels%) at the DNA level of hHAO1-21b in primary human hepatocytes. [Figure 16] 14 depicts the correlation between the % relative indels between different engineered versions of MG29-1 hHAO guide 4b and MG29-1 hHAO1 guide 21b found in Figures 14 and 15. For each guide, the fold improvement compared to chemical 51 is shown. The dashed line indicates the cutoff at 80% relative indels. [Figure 17]

[0023] Figure 1 depicts the editing activity (% indels) of guide cH29-4b-50 in primary cynomolgus monkey hepatocytes. Values are the average of three independent transfections, each performed in triplicate. Error bars indicate the standard deviation among the three transfections.

[0024] Brief Description of Sequence Listing The Sequence Listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions, and systems according to the present disclosure. Below are exemplary descriptions of the sequences therein.

[0025] MG11 SEQ ID NOs: 1 to 37 show the full-length peptide sequences of MG11 nuclease.

[0026] SEQ ID NO: 3471 shows the crRNA 5' direct repeat designed to function with MG11 nuclease.

[0027] SEQ ID NOs: 3472 to 3538 show the effector repeat motif of MG11 nuclease.

[0028] MG13 SEQ ID NOs: 38 to 118 show the full-length peptide sequences of MG13 nuclease.

[0029] SEQ ID NOs: 3540 to 3550 show the effector repeat motif of MG13 nuclease.

[0030] MG19 SEQ ID NOs: 119 to 124 show the full-length peptide sequences of MG19 nuclease.

[0031] SEQ ID NOs: 3551-3558 show the nucleotide sequences of sgRNAs engineered to function with MG19 nuclease.

[0032] SEQ ID NOs: 3863 to 3866 show PAM sequences compatible with MG19 nuclease.

[0033] MG20 SEQ ID NO: 125 shows the full-length peptide sequence of MG20 nuclease.

[0034] SEQ ID NO: 3559 shows the nucleotide sequence of an sgRNA engineered to function with MG20 nuclease.

[0035] SEQ ID NO: 3867 shows a PAM sequence compatible with MG20 nuclease.

[0036] MG26 SEQ ID NOs: 126 to 140 show the full-length peptide sequences of MG26 nuclease.

[0037] SEQ ID NOs: 3560 to 3572 show the effector repeat motif of MG26 nuclease.

[0038] MG28 SEQ ID NOs: 141 to 214 show the full-length peptide sequences of MG28 nuclease.

[0039] SEQ ID NOs: 3573 to 3607 show the effector repeat motif of MG28 nuclease.

[0040] SEQ ID NOs: 3608-3609 show crRNA 5' direct repeats designed to function with MG28 nuclease.

[0041] SEQ ID NOs: 3868 to 3869 show PAM sequences compatible with MG28 nuclease.

[0042] MG29 SEQ ID NOs: 215 to 225, 6340 to 6550, and 6563 to 6565 show the full-length peptide sequences of MG29 nuclease.

[0043] SEQ ID NO: 5680 shows the nucleotide sequence of the MG29-1 nuclease, including the 5'UTR, NLS, CDS, NLS, 3'UTR, and polyA tail.

[0044] SEQ ID NOs: 3610-3611 show the effector repeat motif of MG29 nuclease.

[0045] SEQ ID NO: 3612 shows the nucleotide sequence of an sgRNA engineered to function with MG29 nuclease.

[0046] SEQ ID NOs: 3870 to 3872 show PAM sequences compatible with MG29 nuclease.

[0047] SEQ ID NO: 5687 shows the MG29-1 coding sequence used to produce mRNA.

[0048] SEQ ID NOs: 5830, 5846, and 6582-6588 show DNA sequences encoding MG29-1 mRNA.

[0049] MG30 SEQ ID NOs: 226 to 228 show the full-length peptide sequences of MG30 nuclease.

[0050] SEQ ID NOs: 3613 to 3615 show the effector repeat motif of MG30 nuclease.

[0051] SEQ ID NO: 3873 shows a PAM sequence compatible with MG30 nuclease.

[0052] MG31 SEQ ID NOs: 229 to 260 show the full-length peptide sequences of MG31 nuclease.

[0053] SEQ ID NOs: 3616 to 3632 show the effector repeat motifs of MG31 nuclease.

[0054] SEQ ID NOs: 3874 to 3876 show PAM sequences compatible with MG31 nuclease.

[0055] MG32 SEQ ID NO: 261 shows the full-length peptide sequence of MG32 nuclease.

[0056] SEQ ID NOs: 3633-3634 show the effector repeat motif of MG32 nuclease.

[0057] SEQ ID NO: 3876 shows a PAM sequence compatible with MG32 nuclease.

[0058] MG37 SEQ ID NOs: 262 to 426 show the full-length peptide sequences of MG37 nuclease.

[0059] SEQ ID NO: 3635 shows the effector repeat motif of MG37 nuclease.

[0060] 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, and 3660-3661 show the nucleotide sequences of sgRNAs engineered to function with MG37 nuclease.

[0061] SEQ ID NOs: 3638, 3642, 3646, 3650, 3654, 3658, and 3662 set forth the nucleotide sequence of the MG37 tracrRNA, which is derived from the same locus as the MG37 nuclease described above.

[0062] 3639, 3643, 3647, 3651, 3655, and 3659 indicate 5' direct repeat sequences derived from the native MG37 locus that function as crRNAs when placed 5' to a 3' targeting or spacer sequence.

[0063] MG53 SEQ ID NOs: 427 to 428 show the full-length peptide sequences of MG53 nuclease.

[0064] SEQ ID NO: 3663 shows a 5' direct repeat sequence derived from the native MG53 locus that functions as a crRNA when placed 5' to a 3' targeting or spacer sequence.

[0065] SEQ ID NOs: 3664-3667 show the nucleotide sequences of sgRNAs engineered to function with MG53 nuclease.

[0066] SEQ ID NOs: 3668-3669 show the nucleotide sequence of MG53 tracrRNA, which is derived from the same locus as the MG53 nuclease described above.

[0067] MG54 SEQ ID NOs: 429 to 430 show the full-length peptide sequences of MG54 nuclease.

[0068] SEQ ID NO: 3670 shows a 5' direct repeat sequence derived from the native MG54 locus that functions as a crRNA when placed 5' to a 3' targeting or spacer sequence.

[0069] SEQ ID NOs: 3671-3672 show the nucleotide sequences of sgRNAs engineered to function with MG54 nuclease.

[0070] SEQ ID NOs: 3673-3676 show the nucleotide sequence of MG54 tracrRNA, which is derived from the same locus as the MG54 nuclease described above.

[0071] MG55 SEQ ID NOs: 431 to 688 show the full-length peptide sequences of MG55 nuclease.

[0072] SEQ ID NO: 6031 shows the nucleotide sequence of an sgRNA engineered to function with MG55 nuclease.

[0073] SEQ ID NO: 6032 shows a PAM sequence compatible with MG55 nuclease.

[0074] MG56 SEQ ID NOs: 689 to 690 show the full-length peptide sequences of MG56 nuclease.

[0075] SEQ ID NO: 3678 shows the crRNA 5' direct repeat designed to function with MG56 nuclease.

[0076] SEQ ID NOs: 3679 to 3680 show the effector repeat motif of MG56 nuclease.

[0077] MG57 SEQ ID NOs: 691 to 721 show the full-length peptide sequences of MG57 nuclease.

[0078] SEQ ID NOs: 3681 to 3694 show the effector repeat motif of MG57 nuclease.

[0079] SEQ ID NOs: 3695-3696 show the nucleotide sequences of sgRNAs engineered to function with MG57 nuclease.

[0080] SEQ ID NOs: 3879 to 3880 show PAM sequences compatible with MG57 nuclease.

[0081] MG58 SEQ ID NOs: 722 to 779 show the full-length peptide sequences of MG58 nuclease.

[0082] SEQ ID NOs: 3697 to 3711 show the effector repeat motif of MG58 nuclease.

[0083] MG59 SEQ ID NOs: 780 to 792 show the full-length peptide sequences of MG59 nuclease.

[0084] SEQ ID NOs: 3712 to 3728 show the effector repeat motifs of MG59 nuclease.

[0085] SEQ ID NOs: 3729-3730 show the nucleotide sequences of sgRNAs engineered to function with MG59 nuclease.

[0086] SEQ ID NOs: 3881-3882 show PAM sequences compatible with MG59 nuclease.

[0087] MG60 SEQ ID NOs: 793 to 1163 show the full-length peptide sequences of MG60 nuclease.

[0088] SEQ ID NOs: 3731 to 3733 show the effector repeat motif of MG60 nuclease.

[0089] MG61 SEQ ID NOs: 1164 to 1469 show the full-length peptide sequences of MG61 nuclease.

[0090] SEQ ID NOs: 3734-3735 show crRNA 5' direct repeats designed to function with MG61 nuclease.

[0091] SEQ ID NOs: 3736 to 3847 show the effector repeat motif of MG61 nuclease.

[0092] MG62 SEQ ID NOs: 1470 to 1472 show the full-length peptide sequences of MG62 nuclease.

[0093] SEQ ID NOs: 3848 to 3850 show the effector repeat motif of MG62 nuclease.

[0094] MG70 SEQ ID NOs: 1473 to 1514 show the full-length peptide sequences of MG70 nuclease.

[0095] MG75 SEQ ID NOs: 1515 to 1710 show the full-length peptide sequences of MG75 nuclease.

[0096] MG77 SEQ ID NOs: 1711 to 1712 show the full-length peptide sequences of MG77 nuclease.

[0097] SEQ ID NOs: 3851-3852 show the nucleotide sequences of sgRNAs engineered to function with MG77 nuclease.

[0098] SEQ ID NOs: 3883 to 3884 show PAM sequences compatible with MG77 nuclease.

[0099] MG78 SEQ ID NOs: 1713 to 1717 show the full-length peptide sequences of MG78 nuclease.

[0100] SEQ ID NO: 3853 shows the nucleotide sequence of an sgRNA engineered to function with MG78 nuclease.

[0101] SEQ ID NO: 3885 shows a PAM sequence compatible with MG78 nuclease.

[0102] MG79 SEQ ID NOs: 1718 to 1722 show the full-length peptide sequences of MG79 nuclease.

[0103] SEQ ID NOs: 3854-3857 show the nucleotide sequences of sgRNAs engineered to function with MG79 nuclease.

[0104] SEQ ID NOs: 3886 to 3889 show PAM sequences compatible with MG79 nuclease.

[0105] MG80 SEQ ID NO: 1723 shows the full-length peptide sequence of MG80 nuclease.

[0106] MG81 SEQ ID NOs: 1724 to 2654 show the full-length peptide sequences of MG81 nuclease.

[0107] MG82 SEQ ID NOs: 2655 to 2657 show the full-length peptide sequences of MG82 nuclease.

[0108] MG83 SEQ ID NOs: 2658-2659 show the full-length peptide sequences of MG83 nuclease.

[0109] MG84 SEQ ID NOs: 2660 to 2677 show the full-length peptide sequences of MG84 nuclease.

[0110] MG85 SEQ ID NOs: 2678 to 2680 show the full-length peptide sequences of MG85 nuclease.

[0111] MG90 SEQ ID NOs: 2681 to 2809 show the full-length peptide sequences of MG90 nuclease.

[0112] MG91 SEQ ID NOs: 2810 to 3470 and 6274 to 6281 show the full-length peptide sequences of MG91 nuclease.

[0113] SEQ ID NOs: 6033-6036, 6284-6325, and 6567-6581 set forth the nucleotide sequences of sgRNAs engineered to function with MG91 nuclease.

[0114] SEQ ID NOs: 6040-6049 and 6282 represent the MG91 intergenic region potentially encoding tracrRNA.

[0115] SEQ ID NOs: 6050-6059 and 6283 represent MG91 CRISPR repeats.

[0116] Spacer Segment SEQ ID NOs: 3858 to 3861 show the nucleotide sequences of the spacer segments.

[0117] NLS SEQ ID NOs: 3938-3953 show the sequences of exemplary nuclear localization sequences (NLS) that can be added to nucleases according to the present disclosure.

[0118] CD38 targeting SEQ ID NOs: 4428-4465 and 5685 set forth the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target CD38.

[0119] SEQ ID NOs: 4466 to 4503 and 5686 show the DNA sequences of the CD38 target site.

[0120] TIGIT targeting SEQ ID NOs: 4504-4520 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target TIGIT.

[0121] SEQ ID NOs: 4521 to 4537 show the DNA sequences of the TIGIT target sites.

[0122] AAVS1 targeting SEQ ID NOs: 4538-4568 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target AAVS1.

[0123] SEQ ID NOs: 4569 to 4599 show the DNA sequences of the AAVS1 target sites.

[0124] B2M targeting SEQ ID NOs: 4600-4675 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target B2M.

[0125] SEQ ID NOs: 4676 to 4751 show the DNA sequences of the B2M target sites.

[0126] CD2 targeting SEQ ID NOs: 4752-4836 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target CD2.

[0127] SEQ ID NOs: 4837 to 4921 show the DNA sequences of the CD2 target sites.

[0128] CD5 targeting SEQ ID NOs: 4922-4945 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target CD5.

[0129] SEQ ID NOs: 4946 to 4969 show the DNA sequences of the CD5 target sites.

[0130] hRosa26 targeting SEQ ID NOs: 4970-5012 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target hRosa26.

[0131] SEQ ID NOs: 5013 to 5055 show the DNA sequences of the hRosa26 target sites.

[0132] TRAC targeting SEQ ID NOs: 5056-5125, 5681, and 5683 set forth the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target TRAC.

[0133] SEQ ID NOs: 5126-5195, 5682, and 5684 show the DNA sequences of the TRAC target sites.

[0134] TRBC1 targeting SEQ ID NOs: 5196-5210 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target TRBC1.

[0135] SEQ ID NOs: 5211 to 5225 show the DNA sequences of the TRBC1 target sites.

[0136] TRBC2 targeting SEQ ID NOs: 5226-5246 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target TRBC2.

[0137] SEQ ID NOs: 5247 to 5267 show the DNA sequences of the TRBC2 target sites.

[0138] TRBC1 / 2 targeting SEQ ID NOs: 5642-5660 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target TRBCs.

[0139] SEQ ID NOs: 5661 to 5679 show the DNA sequences of the TRBC target sites.

[0140] FAS targeting SEQ ID NOs: 5268-5366 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target FAS.

[0141] SEQ ID NOs: 5367 to 5465 show the DNA sequences of the FAS target sites.

[0142] PD-1 targeting SEQ ID NOs: 5466-5473 set forth the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target PD-1.

[0143] SEQ ID NOs: 5474 to 5481 show the DNA sequences of the PD-1 target sites.

[0144] HPRT targeting SEQ ID NOs: 5482-5561 set forth the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target HPRT.

[0145] SEQ ID NOs: 5562 to 5641 show the DNA sequences of the HPRT target sites.

[0146] HAO-1 targeting SEQ ID NOs: 5788-5829, 5831-5834, 6909-6930, and 6953 set forth the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human HAO-1.

[0147] SEQ ID NOs: 6931 to 6952 show the DNA sequences of the human HAO-1 target site.

[0148] SEQ ID NO: 6954 shows the nucleotide sequence of an sgRNA engineered to function with MG29-1 nuclease to target cynomolgus monkey HAO-1.

[0149] SEQ ID NOs: 5836-5845 and 6955 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target mouse HAO-1.

[0150] APO-A1 targeting SEQ ID NOs: 5847-5860 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target mouse APO-A1.

[0151] SEQ ID NOs: 5861 to 5874 show the DNA sequences of the APO-A1 target sites.

[0152] Mouse ANGPTL3 targeting SEQ ID NOs: 5875-5952 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target mouse ANGPTL3.

[0153] SEQ ID NOs: 5953 to 6030 show the DNA sequences of mouse ANGPTL3 target sites.

[0154] MG29-1 human GPR146 targeting SEQ ID NOs: 6060-6068 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human GPR146.

[0155] SEQ ID NOs: 6069 to 6077 show the DNA sequences of the human GPR146 target site.

[0156] MG29-1 mouse GPR146 targeting SEQ ID NOs: 6078-6079 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target mouse GPR146.

[0157] SEQ ID NOs: 6080 to 6081 show the DNA sequences of the mouse GPR146 target site.

[0158] Human ANGPTL3 targeting SEQ ID NOs: 6082-6177 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human ANGPTL3.

[0159] SEQ ID NOs: 6178 to 6273 show the DNA sequences of human ANGPTL3 target sites.

[0160] Human VCP targeting SEQ ID NOs: 6551-6556 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human VCP.

[0161] SEQ ID NOs: 6557 to 6562 show the DNA sequences of the human VCP target site. DETAILED DESCRIPTION OF THE INVENTION

[0162] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0163] The practice of some methods disclosed herein employs, unless otherwise indicated, techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds.), the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988), Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0164] 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 the terms "including," "includes," "having," "has," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0165] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends 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 standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0166] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Contemplated nucleotides include naturally occurring and synthetic nucleotides. A nucleotide is a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes the 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, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide encompasses dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of ddNTP include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.Nucleotide can be unlabeled, or can be detectably labeled, for example, by using an optically detectable moiety (e.g., fluorophore) or a moiety containing quantum dot.Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, all available from Perkin Elmer (Foster City, Calif.); FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP available from Boehringer (Mannheim, Indianapolis, Ind.), and Molecular Examples of chromosomal labeled nucleotides available from Probes (Eugene, Oreg.) include 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. The term nucleotide encompasses chemically modified nucleotides. An exemplary chemically modified nucleotide is biotin-dNTP.Non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0167] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in either single-, double-, or multiple-stranded form. Contemplated polynucleotides include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, DNA, RNA, coding or non-coding regions of genes or gene fragments, multiple loci (single locus) defined from binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. When referring to T, in polynucleotides, T refers to U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to a cell and / or can exist in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., modified backbones, sugars, or nucleobases). 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 acids, heterologous nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to the sugar), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0168] The term "transfection" or "transfected" refers to the introduction of a polynucleotide into a cell by non-viral or viral-based methods. The polynucleotide may be a gene sequence encoding an entire protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0169] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by a peptide bond. The term does not denote a specific length of the polymer, and is not intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer is interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structure (e.g., domains). The term also encompasses amino acid polymers that have been modified by, for example, disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" refer to natural and unnatural 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 chemical moiety that does not occur naturally on the amino acid. The term "amino acid" includes both D- and L-amino acids.

[0170] As used herein, "non-naturally occurring" refers to a nucleic acid or polypeptide sequence that does not occur in nature. Non-naturally occurring refers to a nucleic acid or polypeptide sequence that does not occur in nature, including modifications such as mutations, insertions, or deletions. The term non-naturally occurring encompasses fusion nucleic acids or polypeptides in which the non-naturally occurring sequence encodes or exhibits an activity (e.g., an enzymatic activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, a ubiquitination activity, etc.) of the nucleic acid or polypeptide sequence to which it is fused. Non-naturally occurring nucleic acid or polypeptide sequences include those that are linked by genetic engineering to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to produce a chimeric nucleic acid or polypeptide sequence that encodes the chimeric nucleic acid or polypeptide.

[0171] As used herein, the term "promoter" refers to a regulatory DNA region that controls the transcription or expression of a polynucleotide (e.g., a gene) and may be located adjacent to or overlapping the nucleotide or region of nucleotide at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often referred to as transcription factors, that facilitate the binding of RNA polymerase to DNA, resulting in gene transcription. Basal promoters in eukaryotes typically, but not necessarily, contain a TATA box and / or a CAAT box.

[0172] The term "expression," as used herein, refers to the process by which a nucleic acid sequence or 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 a peptide, polypeptide, or protein. Collectively, the transcript and the encoded polypeptide may be referred to as a "gene product." If the polynucleotide is derived from genomic DNA, the term expression includes splicing of mRNA in a eukaryotic cell.

[0173] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents refer to the arrangement of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., such that operation (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element can be, but 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 activation of the second element. A regulatory element, which may include, for example, a promoter and / or enhancer sequence, is operably linked to a coding region if the regulatory element helps to initiate transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region, so long as this functional relationship is maintained.

[0174] As used herein, "vector" refers to a polymer or association of polymers that contains or is associated with a polynucleotide and mediates delivery of the polynucleotide to a cell. Examples of vectors include nucleic acid-based vectors (e.g., plasmids and viral vectors) and liposomes. Exemplary nucleic acid-based vectors generally include genetic elements, e.g., regulatory elements, operably linked to a gene to facilitate expression of the gene in the target.

[0175] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a component of a vector that contains a combination of nucleic acid sequences or elements (e.g., a therapeutic gene, a promoter, and a terminator) that are to be expressed together or that are operably linked for expression. The term encompasses expression cassettes that contain a combination of one or more genes with regulatory elements that are operably linked for expression.

[0176] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes the ability to affect expression in a manner attributable to the full-length sequence.

[0177] The terms "engineered," "synthetic," and "artificial" are used interchangeably herein to refer to entities modified by human intervention. For example, the terms refer to polynucleotides or polypeptides that do not occur in nature. Engineered peptides may, but need not, have low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to naturally occurring human proteins. For example, the VPR domain and the VP64 domain are synthetic transactivation domains. Non-limiting examples include: nucleic acids that are modified by changing their sequence to one that does not occur in nature; nucleic acids that are modified by ligating to a nucleic acid with which they are not naturally associated such that the ligated product possesses a function not present in the original nucleic acid; engineered nucleic acids that are synthesized in vitro using a sequence that does not occur in nature; proteins that are modified by changing their amino acid sequence to a sequence that does not occur in nature; and engineered proteins that acquire new functions or properties. An "engineered" system contains at least one engineered component.

[0178] As used herein, the term "Cas12a" refers to a family of Cas endonucleases that are class 2, type VA Cas endonucleases that (a) use relatively small guide RNAs (approximately 42-44 nucleotides) that are processed by the nuclease itself after transcription from the CRISPR array, and (b) cleave DNA to leave staggered cleavage sites.

[0179] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize to a target nucleic acid, thereby directing an associated nuclease to the target nucleic acid. A guide nucleic acid can be, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. A guide nucleic acid can include crRNA or tracrRNA, or a combination of both. The term guide nucleic acid encompasses engineered guide nucleic acids and programmable guide nucleic acids that specifically bind to a target nucleic acid. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to a guide nucleic acid and hybridizes with the guide nucleic acid is the complementary strand. A strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore not complementary to the guide nucleic acid is referred to as the non-complementary strand. A guide nucleic acid having a polynucleotide strand is a "single guide nucleic acid." A guide nucleic acid having two polynucleotide strands is a "double guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" is inclusive and refers to both single and double guide nucleic acids. A guide nucleic acid may include a segment referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence" or "spacer." A nucleic acid targeting segment may include a subsegment referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment."

[0180] As used herein, the terms "gene editing" and "genome editing" can be used interchangeably. Gene editing or genome editing refers to changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0181] As used herein, the term "complex" refers to the joining of at least two components. Each of the two components may retain the properties / activities it had prior to forming the complex or may acquire properties as a result of forming the complex. Joining may be by, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonding), use of a linker, fusion, or any other suitable method. Contemplated components of the complex include polynucleotides, polypeptides, or combinations thereof. For example, the complex may include an endonuclease and a guide polynucleotide.

[0182] The term "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are identical or have a specified percentage of the same amino acid residues or nucleotides 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, for example, BLASTP using the BLOSUM62 scoring matrix setting parameters of a word length (W) of 3, an expectation (E) of 10, and a gap cost of 11, an extension of 1, and using a conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of a word length (W) of 2, an expectation (E) of 1,000,000, and PAM30 scoring setting gap costs at 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (these are the default parameters for BLASTP in the BLAST suite available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using Smith-Waterman homology search algorithm parameters of 2 matches, -1 mismatches, and -1 gaps; MUSCLE using default parameters; MAFFT using parameters of 2 retries and 1000 maximum repeats; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0183] In the context of two or more nucleic acid or polypeptide sequences, the term "optimally aligned" generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences aligned for maximum amino acid residue or nucleotide correspondence, as determined, for example, by the alignment producing the highest or "optimized" percent identity score.

[0184] Variants of any of the enzymes described herein having one or more conservative amino acid substitutions are included in the present disclosure. 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 achieved by substituting amino acids with similar hydrophobicity, polarity, and R chain length for each other. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by finding amino acid residues (e.g., non-conserved residues) that vary between species without changing the basic function of the encoded protein. Such conservatively substituted variants have at least about 20% similarity to any one of the endonuclease protein sequences described herein (e.g., an MG11, MG13, MG26, MG28, MG29, MG30, MG31, MG32, MG37, MG53, MG54, MG55, MG56, MG57, MG58, MG59, MG60, MG61, MG62, MG70, MG82, MG83, MG84, or MG85 family endonuclease described herein, or any family nuclease described herein). Conservatively substituted variants may include variants having 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% sequence identity. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can include sequences with substitutions of one or more critical active site residues or guide RNA binding residues of the endonuclease such that the activity of the endonuclease is not disrupted. In some embodiments, functional variants of any of the proteins described herein lack substitution of at least one conserved or functional residue.In some embodiments, a functional variant of any of the proteins described herein lacks all conserved or functional residue substitutions.

[0185] The present disclosure also includes variants (e.g., reduced activity variants) of any of the enzymes described herein that have substitutions of one or more catalytic residues to reduce or eliminate the activity of the enzyme. In some embodiments, reduced activity variants of the proteins described herein include at least one, at least two, or all three catalytic residues.

[0186] Conservative substitution tables providing functionally similar amino acids are available in various references (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH 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); and 8) Cysteine (C), methionine (M).

[0187] overview The discovery of new Cas enzymes with unique functionality and structure offers the potential to further improve gene editing technologies, their speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly interspaced short palindromic repeats (CRISPR) systems in microorganisms and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is, in part, because the vast number of microbial species may not be easily cultivated under laboratory conditions. Metagenomic sequencing from natural environmental niches containing large numbers of microbial species could dramatically increase the number of characterized new CRISPR / Cas systems and offer the potential to accelerate the discovery of novel oligonucleotide editing functions. A fruitful recent example of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0188] CRISPR / Cas systems are RNA-directed nuclease complexes that function as adaptive immune systems in microorganisms. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally consist of two parts: (i) an array of short repeat sequences (30–40 bp) separated by short spacer sequences that encode RNA-based targeting elements, and (ii) an ORF encoding a Cas nuclease. Efficient nuclease targeting of a specific target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6–8 nucleic acids of the target nucleic acid and the crRNA guide and (ii) the presence of a protospacer adjacent motif (PAM) sequence within a certain vicinity of the target nucleic acid sequence, depending on the specific Cas nuclease (PAM is usually a sequence not commonly represented in the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are generally organized into two classes, five types, and 16 subtypes based on shared functional characteristics and evolutionary similarities.

[0189] Class 1 CRISPR-Cas systems have large, multi-subunit effector complexes and include Type I, Type III, and Type IV Cas nucleases, while Class 2 CRISPR-Cas systems generally have single polypeptide multi-domain nuclease effectors and include Type II, Type V, and Type VI Cas nucleases.

[0190] Type II CRISPR-Cas systems are considered the simplest in terms of components. In type II CRISPR-Cas systems, processing of the CRISPR array into mature crRNA does not require the presence of a specialized endonuclease subunit, but rather a small transcoded 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 the mature effector enzyme loaded with both the tracrRNA and crRNA. Cas II nucleases are identified as DNA nucleases. Type II effectors generally exhibit a structure containing a RuvC-like endonuclease domain that fits into an RNase H fold with an unrelated HNH nuclease domain inserted within the RuvC-like nuclease domain fold. The RuvC-like domain is responsible for cleavage of the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

[0191] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Cas12) structure similar to that of type II effectors, containing a RuvC-like domain. Like type II, most (if not all) type V CRISPR systems use a tracrRNA to process precursor crRNA into mature crRNA. However, unlike type II systems, which require RNAse III to cleave precursor crRNA into multiple crRNAs, type V systems can cleave precursor crRNA using the effector nuclease itself. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are also identified as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to have robust single-strand nonspecific deoxyribonuclease activity that is activated by the first crRNA-directed cleavage of the double-stranded target sequence.

[0192] CRISPR-Cas systems have recently emerged as a gene editing technology due to their targetability and ease of use. The most commonly used systems are class 2, type II SpCas9 and class 2, type VA Cas12a. Type VA systems are becoming more widely used, particularly because their reported specificity in cells is higher than that of other nucleases, with fewer or no off-target effects. VA systems are also advantageous in that the guide RNA is small (42-44 nucleotides compared to approximately 100 nt for SpCas9) and is processed by the nuclease itself after transcription from the CRISPR array, simplifying multiplexed applications involving multiple gene editing. Furthermore, VA systems have staggered cut sites, which may facilitate directed repair pathways such as microhomology-dependent targeted integration (MITI).

[0193] The most commonly used type VA enzymes require a 5' protospacer adjacent motif (PAM) next to the selected target site: 5'-TTTV-3' for Lachnospiraceae bacterium ND2006 LbCas12a and Acidaminococcus species AsCas12a; and 5'-TTV-3' for Francisella novicida FnCas12a. Recent surveys of orthologs have revealed proteins with less restrictive PAM sequences, such as YTV, YYN, or TTN, that are also active in mammalian cell culture. However, these enzymes do not fully encompass the biodiversity and targetability of VA enzymes and may not represent all possible activities and PAM sequence requirements. Here, thousands of genome fragments from multiple metagenomes have been identified for type VA nucleases. The diversity of identified VA enzymes may be expanded, and improved systems may be developed into highly targetable, compact, and precise gene editing agents.

[0194] MG enzyme In certain embodiments, endonucleases (eg, type V endonucleases) are described herein.

[0195] In some embodiments, the endonuclease is an MG11 nuclease described herein (e.g., SEQ ID NOs: 1-37). In some embodiments, the endonuclease is an MG13 nuclease described herein (e.g., SEQ ID NOs: 38-118). In some embodiments, the endonuclease is an MG19 nuclease described herein (e.g., SEQ ID NOs: 119-124). In some embodiments, the endonuclease is an MG11 nuclease described herein (e.g., SEQ ID NOs: 125). In some embodiments, the endonuclease is an MG26 nuclease described herein (e.g., SEQ ID NOs: 126-140). In some embodiments, the endonuclease is an MG28 nuclease described herein (e.g., SEQ ID NOs: 141-214). In some embodiments, the endonuclease is an MG29 nuclease described herein (e.g., SEQ ID NOs: 215-225, and 6340-6550, and 6563-6565). In some embodiments, the endonuclease is an MG30 nuclease described herein (e.g., SEQ ID NOs: 226-228). In some embodiments, the endonuclease is an MG31 nuclease described herein (e.g., SEQ ID NOs: 229-260). In some embodiments, the endonuclease is an MG32 nuclease described herein (e.g., SEQ ID NOs: 261). In some embodiments, the endonuclease is an MG37 nuclease described herein (e.g., SEQ ID NOs: 262-426). In some embodiments, the endonuclease is an MG53 nuclease described herein (e.g., SEQ ID NOs: 427-428). In some embodiments, the endonuclease is an MG54 nuclease described herein (e.g., SEQ ID NOs: 429-430). In some embodiments, the endonuclease is an MG55 nuclease described herein (e.g., SEQ ID NOs: 431-688). In some embodiments, the endonuclease is an MG56 nuclease described herein (e.g., SEQ ID NOs: 689-690).In some embodiments, the endonuclease is an MG57 nuclease described herein (e.g., SEQ ID NOs: 691-721). In some embodiments, the endonuclease is an MG58 nuclease described herein (e.g., SEQ ID NOs: 722-779). In some embodiments, the endonuclease is an MG59 nuclease described herein (e.g., SEQ ID NOs: 780-792). In some embodiments, the endonuclease is an MG60 nuclease described herein (e.g., SEQ ID NOs: 793-1163). In some embodiments, the endonuclease is an MG61 nuclease described herein (e.g., SEQ ID NOs: 1164-1469). In some embodiments, the endonuclease is an MG62 nuclease described herein (e.g., SEQ ID NOs: 1470-1472). In some embodiments, the endonuclease is an MG70 nuclease described herein (e.g., SEQ ID NOs: 1473-1514). In some embodiments, the endonuclease is an MG75 nuclease described herein (e.g., SEQ ID NOs: 1515-1710). In some embodiments, the endonuclease is an MG77 nuclease described herein (e.g., SEQ ID NOs: 1711-1712). In some embodiments, the endonuclease is an MG78 nuclease described herein (e.g., SEQ ID NOs: 1713-1717). In some embodiments, the endonuclease is an MG79 nuclease described herein (e.g., SEQ ID NOs: 1718-1722). In some embodiments, the endonuclease is an MG80 nuclease described herein (e.g., SEQ ID NO: 1723). In some embodiments, the endonuclease is an MG81 nuclease described herein (e.g., SEQ ID NOs: 1724-2654). In some embodiments, the endonuclease is an MG82 nuclease described herein (e.g., SEQ ID NOs: 2655-2657). In some embodiments, the endonuclease is an MG83 nuclease described herein (e.g., SEQ ID NOs: 2658-2659).In some embodiments, the endonuclease is an MG84 nuclease described herein (e.g., SEQ ID NOs: 2660-2677). In some embodiments, the endonuclease is an MG85 nuclease described herein (e.g., SEQ ID NOs: 2678-2680). In some embodiments, the endonuclease is an MG90 nuclease described herein (e.g., SEQ ID NOs: 2681-2809). In some embodiments, the endonuclease is an MG91 nuclease described herein (e.g., SEQ ID NOs: 2810-3470 and 6274-6281).

[0196] In some embodiments, the endonucleases described herein are about 1000-1100 amino acids in length. In some embodiments, the endonucleases described herein are less than about 400 amino acids in length. In some embodiments, the endonucleases described herein are about 500-700 amino acids in length. In some embodiments, the endonucleases described herein comprise a RuvC and an HTH DNA-binding domain. In some embodiments, the endonucleases comprise a RuvCI, II, or III domain. In some embodiments, the RuvCI domain comprises a D catalytic residue. In some embodiments, the RuvCII domain comprises an E catalytic residue. In some embodiments, the RuvCIII domain comprises a D catalytic residue. In some embodiments, the RuvC domain does not have nuclease activity. In some embodiments, the endonucleases further comprise a WED II domain. In some embodiments, the endonucleases further comprise a zinc finger-like domain. In some embodiments, the endonucleases described herein do not require tracrRNA.

[0197] In some embodiments, the endonuclease is a Cas endonuclease. In some embodiments, the endonuclease is a Class 2, Type V Cas endonuclease. In some embodiments, the endonuclease is a Class 2, Type VA Cas endonuclease. In some embodiments, the endonuclease is not a Cpfl or Cmsl endonuclease. In some embodiments, the endonuclease further comprises a zinc finger-like domain.

[0198] In some embodiments, the endonuclease comprises a sequence having at least about 70% sequence identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease has at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565.In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565.

[0199] In some embodiments, the endonuclease comprises a sequence having at least about 70% sequence identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease has 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: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565.In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 6274-6281, 6340-6550, and 6563-6565.

[0200] In some embodiments, the endonuclease is MG91 endonuclease (i.e., SEQ ID NOs: 6274-6281). In some embodiments, the endonuclease comprises a sequence having at least 70% sequence identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease has 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: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6274-6281.In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 6274-6281.

[0201] In some embodiments, the endonuclease is an MG29 endonuclease (i.e., SEQ ID NOs: 6340-6550 and 6563-6565). In some embodiments, the endonuclease comprises a sequence having at least about 70% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease has 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: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565.In some embodiments, the endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565. In some embodiments, the endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565.

[0202] In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence. In some embodiments, the PAM sequence comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 3863-3913.In some embodiments, the PAM sequence comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 3863-3913. In some embodiments, the PAM sequence comprises a sequence having 100% identity to any one of SEQ ID NOs: 3863-3913.

[0203] In some embodiments, the PAM sequence comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 54 and 96. In some embodiments, the PAM sequence comprises a sequence having at least about 75% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 80% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 85% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 90% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 95% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 96% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 97% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 98% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 99% identity to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having 100% identity to TtTYn, GnYYn, or wCCC.

[0204] In some embodiments, the PAM sequence comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 54 and 96. In some embodiments, the PAM sequence comprises a sequence having at least about 75% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 80% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 85% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 90% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC.In some embodiments, the PAM sequence comprises a sequence having at least about 95% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 96% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 97% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 98% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having at least about 99% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC. In some embodiments, the PAM sequence comprises a sequence having 100% identity to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC.

[0205] In some embodiments, the endonuclease comprises a PI domain having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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%, or 100% sequence identity to the PI (PAM-interacting) domain of any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 54 and 96. In some embodiments, the PI domain comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281.In some embodiments, the PI domain comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281. In some embodiments, the PI domain comprises a sequence having 100% identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281.

[0206] In some embodiments, endonucleases are discovered through metagenomic sequencing. In some embodiments, metagenomic sequencing is performed on samples. In some embodiments, samples are collected from various environments. Such environments may be human microbiomes, animal microbiomes, hot environments, or cold environments. Such environments may include sediments.

[0207] In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises any one of SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises any one of SEQ ID NOs: 3938-3953, 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: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 80% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 85% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 93% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NO: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NO: 3938-3953.In some embodiments, the NLS comprises a sequence having at least about 97% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 98% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 99% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having 100% identity to SEQ ID NOs: 3938-3953.

[0208] [Table 1]

[0209] Guide polynucleotide In certain embodiments, disclosed herein is an engineered endonuclease system comprising: (a) a nuclease disclosed herein; and (b) an engineered guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or dual guide RNAs. When referring to T, in polynucleotides, T means U (uracil) in RNA and T (thymine) in DNA.

[0210] In some embodiments, the engineered guide polynucleotide is configured to form a complex with an engineered endonuclease. In some embodiments, the engineered guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer sequence is configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0211] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, the mammalian cell is a porcine, bovine, caprine, ovine, rodent, rat, mouse, non-human primate, or human cell. In some embodiments, the target gene or target locus is albumin, CD38, TIGIT, AAVS1, B2M, CD2, CD5, hRosa26, TRAC, TRBC1, TRBC2, FAS, PD-1, HPRT, HAO-1, APO-A1, ANGPTL3, GPR146, and VCP.

[0212] In some embodiments, the guide polynucleotide (e.g., gRNA) is selected from the group consisting of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699, 3699-4000, 3699-4001, 3699-4002, 3690-3691, 3691-4003, 3692-3693, 3694-3695, 3695-4004, 3696-3697, 3698-4005, 3699-4006, 3699-4007, 3610-3611, 3612-3613, 3614-3615, 3615-3616, and a sequence having at least 80% sequence identity to the first 19 nucleotides or non-degenerate nucleotides of any one of 71-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide comprises the first 19 nucleotides or non-nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. The degenerate nucleotide sequences include sequences 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 the degenerate nucleotides.In some embodiments, the guide polynucleotide comprises a sequence 100% identical to the first 19 nucleotides or non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.

[0213] In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.

[0214] In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.

[0215] In some embodiments, the target gene is CD38. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4428-4465 and 5685, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4428-4465 and 5685.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4428-4465 and 5685.

[0216] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the CD38 gene or within an intron of the CD38 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4428-4465 and 5685, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685. 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: 4428-4465 and 5685.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: 4428-4465 and 5685. 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: 4428-4465 and 5685.

[0217] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the CD38 gene or within an intron of the CD38 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4466-4503 and 5686, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4466-4503 and 5686. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4466-4503 and 5686.In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4466-4503 and 5686.

[0218] In some embodiments, the target gene is TIGIT. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4504-4520, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4504-4520.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4504-4520.

[0219] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TIGIT gene or within an intron of the TIGIT gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4504-4520, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520. 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: 4504-4520.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: 4504-4520.

[0220] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TIGIT gene or within an intron of the TIGIT gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4521-4537, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4521-4537.

[0221] In some embodiments, the target gene is AAVS1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4538-4568, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4538-4568.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4538-4568.

[0222] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the AAVS1 gene or within an intron of the AAVS1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4538-4568, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568. 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: 4538-4568.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: 4538-4568.

[0223] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the AAVS1 gene or within an intron of the AAVS1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4569-4599, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4569-4599.

[0224] In some embodiments, the target gene is B2M. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4600-4675, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4600-4675.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4600-4675.

[0225] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the B2M gene or within an intron of the B2M gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4600-4675, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675. 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: 4600-4675.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: 4600-4675.

[0226] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the B2M gene or within an intron of the B2M gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4676-4751, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4676-4751.

[0227] In some embodiments, the target gene is CD2. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4752-4836, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4752-4836.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4752-4836.

[0228] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the CD2 gene or within an intron of the CD2 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4752-4836, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836. 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: 4752-4836.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: 4752-4836.

[0229] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the CD2 gene or within an intron of the CD2 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4837-4921, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4837-4921.

[0230] In some embodiments, the target gene is CD5. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4922-4945, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4922-4945.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4922-4945.

[0231] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the CD5 gene or within an intron of the CD5 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4922-4945, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945. 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: 4922-4945.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: 4922-4945.

[0232] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the CD5 gene or within an intron of the CD5 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 4946-4969, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 4946-4969.

[0233] In some embodiments, the target gene is hRosa26. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 4970-5012, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 4970-5012.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 4970-5012.

[0234] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the hRosa26 gene or within an intron of the hRosa26 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 4970-5012, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012. 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: 4970-5012.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: 4970-5012.

[0235] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the hRosa26 gene or within an intron of the hRosa26 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5013-5055, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5013-5055.

[0236] In some embodiments, the target gene is TRAC. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5056-5125, 5681, and 5683, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683.

[0237] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRAC gene or within an intron of the TRAC gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5056-5125, 5681, and 5683, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683.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: 5056-5125, 5681, and 5683. 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: 5056-5125, 5681, and 5683.

[0238] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TRAC gene or within an intron of the TRAC gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5126-5195, 5682, and 5684, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684.In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5126-5195, 5682, and 5684.

[0239] In some embodiments, the target gene is TRBC1 or TRBC2. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660.

[0240] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the TRBC1 or TRBC2 gene or within an intron of the TRBC1 or TRBC2 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660.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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660. 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: 5196-5210, 5226-5246, and 5642-5660.

[0241] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the TRBC1 or TRBC2 gene or within an intron of the TRBC1 or TRBC2 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679.In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679.

[0242] In some embodiments, the target gene is FAS. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5268-5366, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5268-5366.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5268-5366.

[0243] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the FAS gene or within an intron of the FAS gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5268-5366, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366. 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: 5268-5366.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: 5268-5366.

[0244] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the FAS gene or within an intron of the FAS gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5367-5465, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5367-5465.

[0245] In some embodiments, the target gene is PD-1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5466-5473, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5466-5473.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5466-5473.

[0246] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the PD-1 gene or within an intron of the PD-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5466-5473, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473. 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: 5466-5473.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: 5466-5473.

[0247] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the PD-1 gene or within an intron of the PD-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5474-5481, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5474-5481.

[0248] In some embodiments, the target gene is HPRT. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5482-5561, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5482-5561.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5482-5561.

[0249] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HPRT gene or within an intron of the HPRT gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5482-5561, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561. 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: 5482-5561.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: 5482-5561.

[0250] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the HPRT gene or within an intron of the HPRT gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5562-5641, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5562-5641.

[0251] In some embodiments, the target gene is HAO-1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.

[0252] In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 6909-6930 and 6953, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6909-6930 and 6953.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6909-6930 and 6953.

[0253] In some embodiments, the guide polynucleotide is encoded by SEQ ID NO:6954 or SEQ ID NO:6955, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to SEQ ID NO:6954 or SEQ ID NO:6955. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to SEQ ID NO:6954 or SEQ ID NO:6955.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955.

[0254] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955. 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: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.

[0255] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6909-6930 and 6953, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953. 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: 6909-6930 and 6953.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: 6909-6930 and 6953. 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: 6909-6930 and 6953.

[0256] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO:6954 or SEQ ID NO:6955, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955. 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:6954 or SEQ ID NO:6955.In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a sequence having 100% identity to SEQ ID NO:6954 or SEQ ID NO:6955.

[0257] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the HAO-1 gene or within an intron of the HAO-1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 6931-6952, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 6931-6952.

[0258] In some embodiments, the target gene is APO-A1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 5847-5860, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 5847-5860.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 5847-5860.

[0259] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the APO-A1 gene or within an intron of the APO-A1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 5847-5860, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860. 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: 5847-5860.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: 5847-5860.

[0260] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the APO-A1 gene or within an intron of the APO-A1 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 5861-5874, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 5861-5874.

[0261] In some embodiments, the target gene is ANGPTL3. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 6082-6177, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6082-6177.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6082-6177.

[0262] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6082-6177, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177. 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: 6082-6177.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: 6082-6177.

[0263] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 6178-6273, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 6178-6273.

[0264] In some embodiments, the target gene is human GPR146. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 6060-6068, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6060-6068.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6060-6068.

[0265] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6060-6068, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068. 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: 6060-6068.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: 6060-6068.

[0266] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the human GPR146 gene or within an intron of the human GPR146 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 6069-6077, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 6069-6077.

[0267] In some embodiments, the target gene is mouse GPR146. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 6078-6079, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6078-6079.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6078-6079.

[0268] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6078-6079, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079. 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: 6078-6079.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: 6078-6079.

[0269] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 6080-6081, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 6080-6081.

[0270] In some embodiments, the target gene is VCP. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 6551-6556, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous 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: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 6551-6556.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 6551-6556.

[0271] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6551-6556, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556. 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: 6551-6556.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: 6551-6556.

[0272] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the VCP gene or within an intron of the VCP gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 6557-6562, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, a guide polynucleotide hybridizes or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having 100% identity to any one of SEQ ID NOs: 6557-6562.

[0273] In some embodiments, the guide polynucleotide is configured to form a complex with an endonuclease. In some embodiments, the guide polynucleotide binds to the endonuclease to form a complex. In some embodiments, the guide polynucleotide binds to the endonuclease (e.g., non-covalently through electrostatic interactions or hydrogen bonding) to form a complex. In some embodiments, the guide polynucleotide is fused to the endonuclease to form a complex.

[0274] In some embodiments, the guide polynucleotide comprises a spacer sequence. In some embodiments, the spacer sequence is configured to hybridize to the target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0275] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, the mammalian cell is a porcine, bovine, caprine, ovine, rodent, rat, mouse, non-human primate, or human cell.

[0276] In some embodiments, the guide polynucleotide (e.g., guide RNA) comprises various structural elements, including, but not limited to, a spacer sequence that binds to a protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the genome editing system comprises a CRISPR guide RNA. In some embodiments, the guide RNA comprises a crRNA that comprises a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0277] In some embodiments, the systems provided herein comprise one or more guide polynucleotides. In some embodiments, the guide polynucleotide comprises a sense sequence. In some embodiments, the guide polynucleotide comprises an antisense sequence. In some embodiments, the guide polynucleotide comprises a nucleotide sequence other than a region complementary or substantially complementary to a region of the target sequence. For example, a crRNA is part of, or considered part of, the guide polynucleotide, or is included in a guide polynucleotide, e.g., a crRNA:tracrRNA chimera.

[0278] In some embodiments, the guide polynucleotide comprises synthetic or modified nucleotides. In some embodiments, the guide polynucleotide comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, all of the internucleoside linkers of the guide polynucleotide, or the contiguous nucleotide sequence thereof, are modified. For example, in some embodiments, the internucleoside linkages comprise sulfur (S), such as phosphorothioate internucleoside linkages. In some embodiments, the guide polynucleotide comprises more than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified internucleoside linkers (e.g., phosphorothioate internucleoside linkages).

[0279] In some embodiments, a guide polynucleotide comprises a modification to the ribose sugar or nucleobase. In some embodiments, a guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (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 that typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, a sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, a modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0280] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by altering the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, including 2'-substituted nucleosides and LNA (2'-4' biradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification in the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0281] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the guide polynucleotide comprises only modified sugars. In some embodiments, the guide polynucleotide comprises more 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-methyl. In some embodiments, the modified sugar comprises a 2'-fluoro. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising 2'-O-methyl or 2'-fluoro).

[0282] In some embodiments, the guide polynucleotide comprises both internucleoside linker modifications and nucleoside modifications. In some embodiments, the guide polynucleotide comprises more than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers and more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified internucleoside linkers (e.g., phosphorothioate internucleoside linkages) and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified sugars (e.g., comprising 2'-O-methyl or 2'-fluoro).

[0283] In some embodiments, the guide polynucleotide (e.g., gRNA) comprises at least one of the following modifications: (i) a 2'-O methyl or 2'-fluoro base modification of at least one nucleotide within the first four bases of the 5' end of the guide polynucleotide or the last four bases of the 3' end of the guide polynucleotide; (ii) a thiophosphate (PS) linkage between at least two of the first five bases of the 5' end of the guide polynucleotide or a thiophosphate linkage between at least two of the last five bases of the 3' end of the guide polynucleotide; (iii) a thiophosphate linkage within the 3' or 5' stem of the guide polynucleotide; (iv) a 2'-O methyl or 2' base modification within the 3' or 5' stem of the guide polynucleotide; (v) a 2'-fluoro base modification of at least seven bases of the spacer region of the guide polynucleotide; and (vi) a thiophosphate linkage within the loop region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-O methyl or 2'-fluoro base modification of at least one nucleotide within the first five bases of the 5' end of the guide polynucleotide or the last five bases of the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-O methyl or 2'-fluoro base modification at the 5' end of the guide polynucleotide or the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a thiophosphate (PS) linkage between at least two of the first five bases of the 5' end of the guide polynucleotide or a thiophosphate linkage between at least two of the last five bases of the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a thiophosphate linkage within the 3' or 5' stem of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-O methyl base modification within the 3' or 5' stem of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-fluoro base modification of at least seven bases in the spacer region of the guide polynucleotide.In some embodiments, the guide polynucleotide comprises a phosphorothioate bond within the loop region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least three 2'-O-methyl or 2'-fluoro bases at the 5' end of the guide polynucleotide, two phosphorothioate bonds between the first three bases at the 5' end of the guide polynucleotide, at least four 2'-O-methyl or 2'-fluoro bases at the 4' end of the guide polynucleotide, and three phosphorothioate bonds between the last three bases at the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least two 2'-O-methyl bases and at least two phosphorothioate bonds at the 5' end of the guide polynucleotide, and at least one 2'-O-methyl base and at least one phosphorothioate bond at the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one 2'-O-methyl base in both the 3' stem or the 5' stem region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least 1 to at least 14 2'-fluoro bases in the spacer region excluding the seed region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one 2'-O-methyl base in the 5' stem region of the guide polynucleotide and at least 1 to at least 14 2'-fluoro bases in the spacer region excluding the seed region of the guide RNA. In some embodiments, the guide RNA comprises a spacer sequence having at least 80% identity to SEQ ID NO: 3985. In some embodiments, the guide RNA comprises the nucleotides of any one of SEQ ID NOs: 3985-3991, including the chemical modifications listed therein. In some embodiments, the RNA-guided endonuclease is a Cas endonuclease. In some embodiments, the Cas endonuclease is a class 2, type V Cas endonuclease.In some embodiments, the Class 2, Type V Cas endonuclease comprises a RuvC domain, including a RuvCI subdomain, a RuvCII subdomain, and a RuvCIII subdomain. In some embodiments, the Class 2, Type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of 1-3470, 6272-6281, 6340-6550, and 6563-6565, or a variant thereof. In some embodiments, the Class 2, Type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of 141, 215, 229, 261, or 1711-1721, or a variant thereof. In some embodiments, the guide polynucleotide comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, and 3851-3857. In some embodiments, the guide polynucleotide comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3608-3609, 3853, 3851-3857, 6031, 6033-6036, 6284-6325, or 6567-6581.

[0284] In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a plant genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some embodiments, the guide polynucleotide comprises a sequence complementary to a human genomic polynucleotide sequence.

[0285] In some embodiments, the guide polynucleotide is between 30 and 250 nucleotides in length. In some embodiments, the guide polynucleotide is more than 90 nucleotides in length. In some embodiments, 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 polynucleotides are 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, The length is 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.

[0286] In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 8 base pair ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 9 base pair ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 10 base pair ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 11 base pair ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 12 base pair ribonucleotides.

[0287] In some embodiments, the guide polynucleotide comprises a DNA-targeting segment. In some embodiments, the DNA-targeting segment comprises a nucleotide sequence that is complementary to a target sequence. In some embodiments, the target sequence is in a target DNA molecule. In some embodiments, the guide polynucleotide comprises (b) a protein-binding segment. In some embodiments, the protein-binding segment comprises two complementary stretches of nucleotides. In some embodiments, the two complementary stretches of nucleotides hybridize to form a double-stranded RNA (dsRNA) duplex. In some embodiments, the two complementary stretches of nucleotides are covalently linked to each other with an intervening nucleotide.

[0288] In some embodiments, the DNA-targeting segment is positioned 3' to both of the two complementary stretches of nucleotides. In some embodiments, the protein-binding segment comprises a sequence having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the first 19 nucleotides or non-degenerate nucleotides of SEQ ID NO: 3608.

[0289] In some embodiments, the double-stranded RNA (dsRNA) duplex comprises at least 8 ribonucleotides. In some embodiments, the double-stranded RNA (dsRNA) duplex comprises at least 9 ribonucleotides. In some embodiments, the double-stranded RNA (dsRNA) duplex comprises at least 10 ribonucleotides. In some embodiments, the double-stranded RNA (dsRNA) duplex comprises at least 11 ribonucleotides. In some embodiments, the double-stranded RNA (dsRNA) duplex comprises at least 12 ribonucleotides.

[0290] MG endonuclease system In certain embodiments, described herein is an engineered nuclease system comprising an engineered endonuclease and an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

[0291] In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636 and an engineered guide polynucleotide comprising a sequence having about 98% identity to any one of 3637, 3640, 3641, 3644, 3645, 3648, 3649, 3652, 3653, 3656, 3657, 3660, 3661, 3664, 3665, 3671, 3672, 3677, 3678, 3695, 3696, 3729, 3730, 3734, 3735, 3851, 3857, 6031, 6033, 6036, 6284, 6325, and 6567, 6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3642-3643, 3644-3645, 3646-3647, 3648-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699 and an engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having 100% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the guide polynucleotide being any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3651-3659, 3660-3661, 3670-3671, 3680-3681, 3682-3683, 3684-3685, 3686-3687, 3688-3689, 3690-3691, 3692-3693, 3694-3695, 3696-3697, 3698-3699, 3700-3701, 3702-3703, 3704-3705, 3706-3707, 3708-3709, 3710-3711, 3712-3713, 3714-3715, 3716-3717, 3718-3719, 3720-3721, 3722-3723, 3724-3725, 3726-3727, 3728-3729, 3730-3731, and an engineered guide polynucleotide comprising a sequence having 100% identity to any one of: 637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581. In some embodiments, the guide polynucleotide is selected from the group consisting of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, and 3652. a sequence complementary to any one of SEQ ID NOs: 3471, 3539, 3551 to 3559, 3608 to 3609, 3612, 3636 to 3637, 3640 to 3641, 3642 to 3643, 3653, 3656 to 3657, 3660 to 3661, 3664 to 3667, 3671 to 3672, 3677 to 3678, 3695 to 3696, 3729 to 3730, 3734 to 3735, 3851 to 3857, 6031, 6033 to 6036, 6284 to 6325, and 6567 to 6581, 45, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6031, 6033-6036, 6284-6325, and 6567-6581.

[0292] In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to SEQ ID NO: 3612.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to SEQ ID NO: 3612.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to SEQ ID NO: 3612. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having 100% identity to SEQ ID NO: 3612. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 3612 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3612.

[0293] In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the engineered nuclease system comprises: a) an endonuclease comprising a sequence having 100% identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence, the engineered guide polynucleotide comprising a sequence having 100% identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6284-6325 and 6567-6581 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.

[0294] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6909-6930 and 6953.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6909-6930 and 6953.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6909-6930 and 6953.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6909-6930 and 6953, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953.

[0295] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises 100% identity to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NO: 6954 or SEQ ID NO: 6955, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6954 or SEQ ID NO: 6955.

[0296] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6060-6068.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6060-6068.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6060-6068.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6060-6068 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6060-6068.

[0297] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6078-6079.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6078-6079.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6078-6079.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6078-6079.

[0298] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6082-6177.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6082-6177.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 6082-6177 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6082-6177.

[0299] In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 6551-6556.In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 6551-6556. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 6551-6556.In some embod...

Claims

1. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

2. 2. The engineered nuclease system of claim 1, wherein the engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565.

3. 2. The engineered nuclease system of claim 1, wherein the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565.

4. 4. The engineered nuclease system of any one of claims 1 to 3, wherein the engineered guide polynucleotide is a single guide nucleic acid.

5. 4. The engineered nuclease system of any one of claims 1 to 3, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

6. 4. The engineered nuclease system of any one of claims 1 to 3, wherein the engineered guide polynucleotide is RNA.

7. 7. The engineered nuclease system of any one of claims 1 to 6, wherein the engineered endonuclease is non-covalently bound to the engineered guide polynucleotide.

8. 7. The engineered nuclease system of any one of claims 1 to 6, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

9. 7. The engineered nuclease system of any one of claims 1 to 6, wherein the endonuclease is fused to the engineered guide polynucleotide.

10. 10. The engineered nuclease system of any one of claims 1 to 9, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 3612.

11. 10. The engineered nuclease system of any one of claims 1 to 9, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to SEQ ID NO: 3612.

12. 12. The engineered nuclease system of any one of claims 1 to 11, wherein the engineered endonuclease is configured to bind to a PAM comprising any one of SEQ ID NOs: 3870-3872.

13. 12. The engineered nuclease system of any one of claims 1 to 11, wherein the engineered endonuclease is configured to bind to a YYn-containing PAM.

14. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6274-6281; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

15. 15. The engineered nuclease system of claim 14, wherein the engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6274-6281.

16. 15. The engineered nuclease system of claim 14, wherein the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6274-6281.

17. 17. The engineered nuclease system of any one of claims 14 to 16, wherein the engineered guide polynucleotide is a single guide nucleic acid.

18. 17. The engineered nuclease system of any one of claims 14 to 16, wherein the engineered guide polynucleotide is a dual guide nucleic acid.

19. 17. The engineered nuclease system of any one of claims 14 to 16, wherein the engineered guide polynucleotide is RNA.

20. 20. The engineered nuclease system of any one of claims 14 to 19, wherein the engineered endonuclease is non-covalently bound to the engineered guide polynucleotide.

21. 20. The engineered nuclease system of any one of claims 14 to 19, wherein the endonuclease is covalently linked to the engineered guide polynucleotide.

22. 20. The engineered nuclease system of any one of claims 14 to 19, wherein the endonuclease is fused to the engineered guide polynucleotide.

23. 23. The engineered nuclease system of any one of claims 14-22, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.

24. 23. The engineered nuclease system of any one of claims 14 to 22, wherein the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 6284-6325 and 6567-6581.

25. 25. The engineered nuclease system of any one of claims 14 to 24, wherein the engineered endonuclease is configured to bind to a PAM comprising any one of the following sequences: tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC.

26. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953.

27. 27. The engineered nuclease system of claim 26, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6931-6952.

28. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with said endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of said human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068.

29. 29. The engineered nuclease system of Claim 28, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6069-6077.

30. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with said endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of said mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079.

31. 31. The engineered nuclease system of claim 30, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6080-6081.

32. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with said endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of said ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177.

33. 33. The engineered nuclease system of claim 32, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6178-6273.

34. 1. An engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6551-6556.

35. 35. The engineered nuclease system of claim 34, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6557-6562.

36. 36. The engineered nuclease system of any one of claims 1 to 35, wherein the endonuclease comprises at least one of the following mutations when the sequence of the endonuclease is optimally aligned with SEQ ID NO:215: S168R, E172R, N577R, or Y170R.

37. 36. The engineered nuclease system of any one of claims 1 to 35, further comprising a single-stranded or double-stranded DNA repair template comprising, from 5' to 3', a first homologous arm comprising a sequence of at least 20 nucleotides that is 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homologous arm comprising a sequence of at least 20 nucleotides that is 3' to the target sequence.

38. 38. The engineered nuclease system of Claim 37, wherein the first or second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.

39. 39. The engineered nuclease system of claim 37 or 38, wherein the first and second homologous arms are homologous to a prokaryotic, bacterial, fungal, or eukaryotic genomic sequence.

40. 40. A method for modifying a target nucleic acid sequence, the method comprising contacting said target nucleic acid sequence with the engineered nuclease system of any one of claims 1 to 39.

41. 41. The method of claim 40, wherein modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence.

42. 42. The method of claim 40 or 41, wherein the target nucleic acid sequence is within CD38, TIGIT, AAVS1, B2M, CD2, CD5, hRosa26, TRAC, TRBC1, TRBC2, FAS, PD-1, HPRT, HAO-1, APO-A1, ANGPTL3, GPR146, or VCP.

43. 43. The method of any one of claims 40 to 42, wherein the target nucleic acid sequence comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA.

44. The method of any one of claims 40 to 43, wherein the modification is in vitro.

45. The method of any one of claims 40 to 43, wherein the modification is in vivo.

46. The method of any one of claims 40 to 43, wherein the modification is ex vivo.

47. 40. A method of modifying a target nucleic acid sequence in a mammalian cell, the method comprising contacting said mammalian cell with the engineered nuclease system of any one of claims 1 to 39.

48. 48. The method of claim 47, further comprising selecting cells containing said modification.

49. 1. A method for modifying an HAO-1 gene, comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) contacting the HAO-1 gene with an engineered nuclease system comprising: an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the HAO-1 gene or within an intron of the HAO-1 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953.

50. 50. The method of claim 49, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6931-6952.

51. 1. A method for modifying the human GPR146 gene, comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) contacting said human GPR146 gene with an engineered nuclease system comprising: an engineered guide polynucleotide configured to form a complex with said endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within said human GPR146 gene or within an intron of said human GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068.

52. 52. The method of claim 51, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6069-6077.

53. 1. A method for modifying a mouse GPR146 gene, comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) contacting said mouse GPR146 gene with an engineered nuclease system comprising: an engineered guide polynucleotide configured to form a complex with said endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the mouse GPR146 gene or within an intron of said mouse GPR146 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079.

54. 54. The method of claim 53, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6080-6081.

55. 1. A method for modifying an ANGPTL3 gene, comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the ANGPTL3 gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177.

56. 56. The method of claim 55, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6178-6273.

57. 1. A method for modifying a VCP gene, comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6340-6550 and 6563-6565; b) contacting the VCP gene with an engineered nuclease system comprising: an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6551-6556.

58. 58. The method of claim 57, wherein the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6557-6562.

59. A cell comprising the engineered nuclease system of any one of claims 1 to 39.

60. 60. The cell of claim 59, wherein the cell is a eukaryotic cell.

61. 60. The cell of claim 59, wherein the cell is a mammalian cell.

62. 60. The cell of claim 59, wherein the cell is an immortalized cell.

63. 60. The cell of claim 59, wherein the cell is an insect cell.

64. 60. The cell of claim 59, wherein the cell is a yeast cell.

65. 60. The cell of claim 59, wherein the cell is a plant cell.

66. 60. The cell of claim 59, wherein the cell is a fungal cell.

67. 60. The cell of claim 59, wherein the cell is a prokaryotic cell.

68. 60. The cell of claim 59, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

69. 60. The cell of claim 59, wherein the cell is an engineered cell.

70. 60. The cell of claim 59, wherein the cell is a stable cell.

71. 60. The cell of claim 59, wherein the cell is a T cell.

72. 60. The cell of claim 59, wherein the cell is a hematopoietic cell.

73. A lipid nanoparticle, comprising: (a) an engineered nuclease system according to any one of claims 1 to 39; and (b) a cationic lipid; and (c) a sterol; (d) a neutral lipid; and (e) a PEG-modified lipid.

74. The lipid nanoparticle of claim 73, wherein the cationic lipid comprises C12-200, the sterol comprises cholesterol, the neutral lipid comprises DOPE, or the PEG-modified lipid comprises DMG-PEG2000.

75. The lipid nanoparticle of claim 73, wherein the cationic lipid comprises 98N12-5 (TETA5-LAP), DLin DMA, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, or C12-200.

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