Class II V-type CRISPR system

Abstract

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

Description

[0001] Cross-reference to related applications

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

[0003] Cas enzymes and their associated clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs appear to be universal (~45% of bacteria, ~84% of archaea) components of the prokaryotic immune system, used to protect such microorganisms from non-self nucleic acids, such as infectious viruses and plasmids, through CRISPR-RNA-guided nucleic acid cleavage. While the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse, containing a variety of nucleic acid interaction domains. Although CRISPR DNA elements were observed as early as 1987, the programmable endonuclease cleavage capabilities of the CRISPR / Cas complex were not recognized until recently, leading to the use of recombinant CRISPR / Cas systems in various DNA manipulation and gene editing applications.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated 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

[0006] In certain embodiments, an engineered nuclease system is described herein, 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 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 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.

[0007] In certain embodiments, an engineered nuclease system is described herein, 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 a sequence of any one of tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gnGY, mCm, or ryCC.

[0008] In certain embodiments, an engineered nuclease system is described herein, 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 with at least a portion of a target nucleic acid sequence within a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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.

[0009] In certain embodiments, an engineered nuclease system is described herein, 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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.

[0010] 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 comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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.

[0011] 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 comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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.

[0012] 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 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, the engineered guide polynucleotide comprising 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, when the sequence of the endonuclease is optimally aligned with SEQ ID NO: 215, the endonuclease comprises at least one of S168R, E172R, N577R or Y170R mutations. In some embodiments, the engineered nuclease system further comprises a single-stranded or double-stranded DNA repair template, which comprises from 5' to 3': a first homology arm, the first homology arm comprising a sequence of at least 20 nucleotides located at 5' of the target deoxyribonucleic acid sequence; a synthetic DNA sequence of at least 10 nucleotides; and a second homology arm, the second homology arm comprising a sequence of at least 20 nucleotides located at 3' of the target sequence. In some embodiments, the first homology arm or the second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500 or 1,000 nucleotides. In some embodiments, the first homology arm and the second homology arm are homologous to the genomic sequence of prokaryotes, bacteria, fungi or eukaryotes.

[0013] In certain embodiments, described herein is a method for modifying a target nucleic acid sequence, the method 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 to the target nucleic acid sequence, nicking the target nucleic acid sequence, or cutting the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is in 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.

[0014] In certain embodiments, described herein are methods for modifying a target nucleic acid sequence in a mammalian cell, the method comprising contacting the mammalian cell using an engineered nuclease system as described herein. In some embodiments, the method further comprises selecting a cell comprising the modification.

[0015] In certain embodiments, described herein are methods of modifying a HAO-1 gene, the methods comprising contacting the HAO-1 gene using 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, the engineered guide polynucleotide comprising 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.

[0016] In certain embodiments, described herein are methods of modifying a human GPR146 gene, the methods comprising contacting the human GPR146 gene using 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, the engineered guide polynucleotide comprising 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.

[0017] In certain embodiments, described herein are methods of modifying a mouse GPR146 gene, the methods comprising contacting the mouse GPR146 gene using 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, the engineered guide polynucleotide comprising 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.

[0018] In certain embodiments, described herein are methods of modifying an ANGPTL3 gene, the methods comprising contacting the ANGPTL3 gene using 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, the engineered guide polynucleotide comprising 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.

[0019] In certain embodiments, described herein are methods of modifying a VCP gene, the methods comprising contacting the VCP gene using 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, the engineered guide polynucleotide comprising 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.

[0020] In certain embodiments, cells comprising the engineered nuclease systems described herein are 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 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.

[0021] In certain embodiments, lipid nanoparticles are described herein, 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-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, or C12-200.

[0022] Other aspects and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described in the detailed description. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative rather than restrictive in nature. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present disclosure are utilized, and in which:

[0024] Figure 1 Depicted are the results of gene editing of human GPR146 at the DNA level in Hep3B cells.

[0025] Figure 2 Depicted are the results of gene editing of mouse GPR146 at the DNA level in Hepa1-6 cells.

[0026] Figure 3 Depicted are the results of gene editing of human ANGPTL3 at the DNA level in Hep3B cells.

[0027] Figure 4 Depicted are the results of gene editing of human GPR146 at the DNA level in primary human hepatocytes.

[0028] Figure 5 Depicted are the results of gene editing of mouse GPR146 at the DNA level in primary mouse hepatocytes.

[0029] Figures 6A-6C Predicted folds of single guide RNA (sgRNA) sequences without spacers are depicted. TracrRNA and repeat sequences are looped with a GAAA tetraloop. The repeat-antirepeat fold is located at the 3' end of each structure (right circle). Fig. 6A The tracrRNA sequences of these two candidates were obtained from in silico analysis of the intergenic regions, indicating that they may encode tracrRNA. Figure 6B and 6C , tracrRNAs are predicted using a covariance model constructed from previously active sgRNAs. The sgRNA numbers (sg numbers) are shown below the nuclease numbers.

[0030] Fig. 7A and 7B In vitro cleavage assay amplification products are depicted. Fig. 7A A 2% agarose gel with a low molecular weight DNA ladder in the leftmost lane is depicted. Figure 7B Digital gels are depicted. The resulting amplicon product was 188 bp when using a guide carrying a U67 spacer, or 205 bp when using a guide carrying a U40 spacer. The specific sgRNA (sg number) and spacer are shown at the top of the lane for each nuclease.

[0031] Fig. 8A and 8BDepicts the template chain ( Fig. 8A ) and non-template strands ( Figure 8B ) Sequence logo of the protospacer adjacent motif (PAM) sequence obtained by NGS sequencing of the amplified cleavage site on ). The specific sgRNA (sg number) and spacer are shown next to the nuclease number.

[0032] Fig. 9A and 9B Depicts the mapping to the template chain ( Fig. 9A ) and non-template strands ( Fig. 9B ) Histogram of the number of DNA reads for the amplified cleavage site of each MG91 nuclease. The specific sgRNA (sg number) and spacer are shown next to the nuclease number.

[0033] Fig.10 Depicted are the results of gene editing of human VCP at the DNA level in K562 cells.

[0034] Figures 11A-11E Depicted are exemplary purifications of MG91-666 and activity analysis of MG91-666 and MG91-155. Fig.11A An exemplary SDS-PAGE gel monitoring stages of the MG91-666 protein expression induction and purification process is depicted. The expected protein MW is approximately 106 kDa. Fig. 11B Depicted is a chromatogram of a concentrated protein fused to MBP run on an anion exchange column. Peak fractions were collected and concentrated (shaded box). Fig. 11C Depicted is a chromatogram in which the MBP-fused concentrated protein was cleaved after anion exchange to remove the MBP and run on a 200 Increase 10 / 300G size exclusion chromatography column. Peak fractions were collected and concentrated (shaded boxes). Fig.11D Depicted are protein activities assessed in an in vitro cleavage reaction using 521 bp linear DNA as substrate. Lanes: (1) ladder, (2) substrate only, (3) substrate + 20x molar excess RNP. Fig.11E Depicted are protein activities assessed in an in vitro cleavage reaction using 2,281 bp plasmid DNA as substrate. Lanes: (1) ladder, (2) APO (unprimed) protein, (3) substrate + 20x molar excess RNP.

[0035] Figures 12A-12B In vitro cleavage assays with the MG29 ancestral nuclease are depicted. The cleavage activity of the novel type V effectors MG29-229, MG29-230, and MG29-231 were determined by a PAM enrichment protocol. Fig. 12AEffectors expressed by in vitro transcription / translation (IVTT) reactions and added to PAM libraries (dsDNA targets) in the presence of crRNA from active nuclease MG29-1 are described. Cleavage products are amplified by ligation to the cleavage site and subsequent PCR amplification. Fig. 12B Depicts the sequence logo of the preferred PAM sequence and relative to the Fig. 12A The cleavage position of the PAM sequence generated by NGS sequencing of the band identified in MG29-229 is weak and the signal is too low to determine the PAM with a certain degree of confidence by NGS.

[0036] Fig.13 Depicted are the relative effects of a single 2'OMe at each position in the 20 nt spacer of the MG29-1 guide.

[0037] Fig.14 Depicted are analyses of gene editing results (% indel) of hHAO1-4b at the DNA level in primary human hepatocytes.

[0038] Fig.15 Depicted is the analysis of gene editing results (% indel) of hHAO1-21b at the DNA level in primary human hepatocytes.

[0039] Fig.16 Depicted Fig.14 and 15 Correlation between relative indel % between different engineered versions of MG29-1 hHAO guide 4b and MG29-1 hHAO1 guide 21b found in . For each guide, the fold improvement compared to chemical composition 51 is indicated. The dashed line indicates the cutoff at 80% relative indel.

[0040] Fig.17 Depicted are the editing activity (% indel) of the guide cH29-4b-50 in primary cynomolgus monkey hepatocytes. Values ​​are the mean of three independent transfections, each performed in triplicate. Error bars indicate the standard deviation between the three transfections.

[0041] Brief Description of Sequence Listing

[0042] 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 is an exemplary description of the sequences therein.

[0043] MG11

[0044] SEQ ID NOs: 1-37 show the full-length peptide sequence of MG11 nuclease.

[0045] SEQ ID NO:3471 shows a crRNA 5' direct repeat sequence designed to function with the MG11 nuclease.

[0046] SEQ ID NOs:3472-3538 show the effector repeat motif of the MG11 nuclease.

[0047] MG13

[0048] SEQ ID NOs: 38-118 show the full-length peptide sequences of MG13 nuclease.

[0049] SEQ ID NOs:3540-3550 show the effector repeat motif of the MG13 nuclease.

[0050] MG19

[0051] SEQ ID NOs: 119-124 show the full-length peptide sequences of MG19 nuclease.

[0052] SEQ ID NOs:3551-3558 show the nucleotide sequences of sgRNAs engineered to function with the MG19 nuclease.

[0053] SEQ ID NOs: 3863-3866 show PAM sequences compatible with MG19 nuclease.

[0054] MG20

[0055] SEQ ID NO: 125 shows the full-length peptide sequence of MG20 nuclease.

[0056] SEQ ID NO:3559 shows the nucleotide sequence of an sgRNA engineered to function with the MG20 nuclease.

[0057] SEQ ID NO:3867 shows a PAM sequence compatible with MG20 nuclease.

[0058] MG26

[0059] SEQ ID NOs: 126-140 show the full-length peptide sequences of MG26 nuclease.

[0060] SEQ ID NOs:3560-3572 show the effector repeat motif of MG26 nuclease.

[0061] MG28

[0062] SEQ ID NOs: 141-214 show the full-length peptide sequences of MG28 nuclease.

[0063] SEQ ID NOs:3573-3607 show the effector repeat motif of MG28 nuclease.

[0064] SEQ ID NOs:3608-3609 show crRNA 5' direct repeat sequences designed to function with the MG28 nuclease.

[0065] SEQ ID NOs:3868-3869 show PAM sequences compatible with MG28 nuclease.

[0066] MG29

[0067] SEQ ID NOs: 215-225, 6340-6550 and 6563-6565 show the full-length peptide sequence of MG29 nuclease.

[0068] SEQ ID NO:5680 shows the nucleotide sequence of MG29-1 nuclease containing 5'UTR, NLS, CDS, NLS, 3'UTR and polyA tail.

[0069] SEQ ID NOs:3610-3611 show the effector repeat motif of MG29 nuclease.

[0070] SEQ ID NO:3612 shows the nucleotide sequence of an sgRNA engineered to function with the MG29 nuclease.

[0071] SEQ ID NOs:3870-3872 show PAM sequences compatible with MG29 nuclease.

[0072] SEQ ID NO:5687 shows the MG29-1 coding sequence used to generate mRNA.

[0073] SEQ ID NOs: 5830, 5846 and 6582-6588 show the DNA sequence encoding MG29-1 mRNA.

[0074] MG30

[0075] SEQ ID NOs: 226-228 show the full-length peptide sequence of MG30 nuclease.

[0076] SEQ ID NOs:3613-3615 show the effector repeat motif of the MG30 nuclease.

[0077] SEQ ID NO:3873 shows a PAM sequence compatible with MG30 nuclease.

[0078] MG31

[0079] SEQ ID NOs: 229-260 show the full-length peptide sequence of MG31 nuclease.

[0080] SEQ ID NOs:3616-3632 show the effector repeat motif of the MG31 nuclease.

[0081] SEQ ID NOs:3874-3876 show PAM sequences compatible with MG31 nuclease.

[0082] MG32

[0083] SEQ ID NO: 261 shows the full-length peptide sequence of MG32 nuclease.

[0084] SEQ ID NOs:3633-3634 show the effector repeat motif of the MG32 nuclease.

[0085] SEQ ID NO:3876 shows a PAM sequence compatible with MG32 nuclease.

[0086] MG37

[0087] SEQ ID NOs: 262-426 show the full-length peptide sequence of MG37 nuclease.

[0088] SEQ ID NO:3635 shows the effector repeat motif of MG37 nuclease.

[0089] SEQ ID NOs: 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 the MG37 nuclease.

[0090] SEQ ID NOs: 3638, 3642, 3646, 3650, 3654, 3658 and 3662 show the nucleotide sequences of MG37 tracrRNA derived from the same locus as the MG37 nuclease described above.

[0091] SEQ ID NOs: 3639, 3643, 3647, 3651, 3655 and 3659 show 5' direct repeat sequences derived from the native MG37 locus that function as crRNAs when placed 5' to a 3' targeting or spacer sequence.

[0092] MG53

[0093] SEQ ID NOs:427-428 show the full-length peptide sequence of MG53 nuclease.

[0094] SEQ ID NO: 3663 shows a 5' direct repeat sequence derived from the native MG53 locus that acts as a crRNA when placed 5' to a 3' targeting or spacer sequence.

[0095] SEQ ID NOs:3664-3667 show the nucleotide sequences of sgRNAs engineered to function with the MG53 nuclease.

[0096] SEQ ID NOs: 3668-3669 show the nucleotide sequence of the MG53 tracrRNA derived from the same locus as the MG53 nuclease described above.

[0097] MG54

[0098] SEQ ID NOs:429-430 show the full-length peptide sequence of MG54 nuclease.

[0099] 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.

[0100] SEQ ID NOs:3671-3672 show the nucleotide sequences of sgRNAs engineered to function with the MG54 nuclease.

[0101] SEQ ID NOs: 3673-3676 show the nucleotide sequence of MG54 tracrRNA derived from the same locus as the MG54 nuclease described above.

[0102] MG55

[0103] SEQ ID NOs:431-688 show the full-length peptide sequence of MG55 nuclease.

[0104] SEQ ID NO:6031 shows the nucleotide sequence of an sgRNA engineered to function with the MG55 nuclease.

[0105] SEQ ID NO:6032 shows a PAM sequence compatible with MG55 nuclease.

[0106] MG56

[0107] SEQ ID NOs:689-690 show the full-length peptide sequence of MG56 nuclease.

[0108] SEQ ID NO:3678 shows a crRNA 5' direct repeat sequence designed to function with the MG56 nuclease.

[0109] SEQ ID NOs:3679-3680 show the effector repeat motif of the MG56 nuclease.

[0110] MG57

[0111] SEQ ID NOs:691-721 show the full-length peptide sequence of MG57 nuclease.

[0112] SEQ ID NOs:3681-3694 show the effector repeat motif of the MG57 nuclease.

[0113] SEQ ID NOs:3695-3696 show the nucleotide sequences of sgRNAs engineered to function with the MG57 nuclease.

[0114] SEQ ID NOs:3879-3880 show PAM sequences compatible with MG57 nuclease.

[0115] MG58

[0116] SEQ ID NOs:722-779 show the full-length peptide sequence of MG58 nuclease.

[0117] SEQ ID NOs:3697-3711 show the effector repeat sequence motif of MG58 nuclease.

[0118] MG59

[0119] SEQ ID NOs:780-792 show the full-length peptide sequence of MG59 nuclease.

[0120] SEQ ID NOs:3712-3728 show the effector repeat motif of the MG59 nuclease.

[0121] SEQ ID NOs:3729-3730 show the nucleotide sequences of sgRNAs engineered to function with the MG59 nuclease.

[0122] SEQ ID NOs:3881-3882 show PAM sequences compatible with MG59 nuclease.

[0123] MG60

[0124] SEQ ID NOs:793-1163 show the full-length peptide sequence of MG60 nuclease.

[0125] SEQ ID NOs:3731-3733 show the effector repeat motif of MG60 nuclease.

[0126] MG61

[0127] SEQ ID NOs: 1164-1469 show the full-length peptide sequence of MG61 nuclease.

[0128] SEQ ID NOs:3734-3735 show crRNA 5' direct repeat sequences designed to function with the MG61 nuclease.

[0129] SEQ ID NOs:3736-3847 show the effector repeat motif of the MG61 nuclease.

[0130] MG62

[0131] SEQ ID NOs: 1470-1472 show the full-length peptide sequence of MG62 nuclease.

[0132] SEQ ID NOs:3848-3850 show the effector repeat motif of the MG62 nuclease.

[0133] MG70

[0134] SEQ ID NOs: 1473-1514 show the full-length peptide sequence of MG70 nuclease.

[0135] MG75

[0136] SEQ ID NOs: 1515-1710 show the full-length peptide sequence of MG75 nuclease.

[0137] MG77

[0138] SEQ ID NOs: 1711-1712 show the full-length peptide sequence of MG77 nuclease.

[0139] SEQ ID NOs:3851-3852 show the nucleotide sequences of sgRNAs engineered to function with the MG77 nuclease.

[0140] SEQ ID NOs:3883-3884 show PAM sequences compatible with MG77 nuclease.

[0141] MG78

[0142] SEQ ID NOs: 1713-1717 show the full-length peptide sequence of MG78 nuclease.

[0143] SEQ ID NO:3853 shows the nucleotide sequence of an sgRNA engineered to function with the MG78 nuclease.

[0144] SEQ ID NO:3885 shows a PAM sequence compatible with MG78 nuclease.

[0145] MG79

[0146] SEQ ID NOs: 1718-1722 show the full-length peptide sequence of MG79 nuclease.

[0147] SEQ ID NOs:3854-3857 show the nucleotide sequences of sgRNAs engineered to function with the MG79 nuclease.

[0148] SEQ ID NOs:3886-3889 show PAM sequences compatible with MG79 nuclease.

[0149] MG80

[0150] SEQ ID NO: 1723 shows the full-length peptide sequence of MG80 nuclease.

[0151] MG81

[0152] SEQ ID NOs: 1724-2654 show the full-length peptide sequence of MG81 nuclease.

[0153] MG82

[0154] SEQ ID NOs: 2655-2657 show the full-length peptide sequence of MG82 nuclease.

[0155] MG83

[0156] SEQ ID NOs: 2658-2659 show the full-length peptide sequence of MG83 nuclease.

[0157] MG84

[0158] SEQ ID NOs: 2660-2677 show the full-length peptide sequence of MG84 nuclease.

[0159] MG85

[0160] SEQ ID NOs: 2678-2680 show the full-length peptide sequence of MG85 nuclease.

[0161] MG90

[0162] SEQ ID NOs: 2681-2809 show the full-length peptide sequence of MG90 nuclease.

[0163] MG91

[0164] SEQ ID NOs: 2810-3470 and 6274-6281 show the full-length peptide sequence of MG91 nuclease.

[0165] SEQ ID NOs: 6033-6036, 6284-6325, and 6567-6581 show the nucleotide sequences of sgRNAs engineered to function with the MG91 chimeric nuclease.

[0166] SEQ ID NOs: 6040-6049 and 6282 show the MG91 intergenic region potentially encoding tracrRNA.

[0167] SEQ ID NOs:6050-6059 and 6283 show the MG91 CRISPR repeat sequences.

[0168] Spacer segment

[0169] SEQ ID NOs:3858-3861 show the nucleotide sequences of the spacer segments.

[0170] NLS

[0171] SEQ ID NOs: 3938-3953 show the sequences of example nuclear localization sequences (NLS) that can be appended to nucleases according to the present disclosure.

[0172] CD38 targeting

[0173] SEQ ID NOs: 4428-4465 and 5685 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target CD38.

[0174] SEQ ID NOs: 4466-4503 and 5686 show the DNA sequences of the CD38 target sites.

[0175] TIGIT targeting

[0176] SEQ ID NOs:4504-4520 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target TIGIT.

[0177] SEQ ID NOs:4521-4537 show the DNA sequences of the TIGIT target sites.

[0178] AAVS1 targeting

[0179] SEQ ID NOs:4538-4568 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target AAVS1.

[0180] SEQ ID NOs:4569-4599 show the DNA sequences of the AAVS1 target sites.

[0181] B2M Targeting

[0182] SEQ ID NOs:4600-4675 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target B2M.

[0183] SEQ ID NOs:4676-4751 show the DNA sequences of the B2M target sites.

[0184] CD2 targeting

[0185] SEQ ID NOs:4752-4836 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target CD2.

[0186] SEQ ID NOs: 4837-4921 show the DNA sequences of the CD2 target sites.

[0187] CD5 targeting

[0188] SEQ ID NOs:4922-4945 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target CD5.

[0189] SEQ ID NOs:4946-4969 show the DNA sequences of the CD5 target sites.

[0190] hRosa26 targeting

[0191] SEQ ID NOs:4970-5012 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target hRosa26.

[0192] SEQ ID NOs: 5013-5055 show the DNA sequence of the hRosa26 target site.

[0193] TRAC targeting

[0194] SEQ ID NOs: 5056-5125, 5681 and 5683 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target TRAC.

[0195] SEQ ID NOs: 5126-5195, 5682 and 5684 show the DNA sequences of the TRAC target sites.

[0196] TRBC1 targeting

[0197] SEQ ID NOs: 5196-5210 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target TRBC1.

[0198] SEQ ID NOs: 5211-5225 show the DNA sequences of the TRBC1 target sites.

[0199] TRBC2 targeting

[0200] SEQ ID NOs: 5226-5246 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target TRBC2.

[0201] SEQ ID NOs: 5247-5267 show the DNA sequences of the TRBC2 target sites.

[0202] TRBC1 / 2 targeting

[0203] SEQ ID NOs:5642-5660 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target TRBC.

[0204] SEQ ID NOs: 5661-5679 show the DNA sequences of the TRBC target sites.

[0205] FAS targeting

[0206] SEQ ID NOs:5268-5366 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target FAS.

[0207] SEQ ID NOs: 5367-5465 show the DNA sequences of the FAS target sites.

[0208] PD-1 targeting

[0209] SEQ ID NOs:5466-5473 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target PD-1.

[0210] SEQ ID NOs: 5474-5481 show the DNA sequences of the PD-1 target sites.

[0211] HPRT Targeting

[0212] SEQ ID NOs:5482-5561 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target HPRT.

[0213] SEQ ID NOs:5562-5641 show the DNA sequence of the HPRT target site.

[0214] HAO-1 targeting

[0215] SEQ ID NOs: 5788-5829, 5831-5834, 6909-6930 and 6953 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human HAO-1.

[0216] SEQ ID NOs: 6931-6952 show the DNA sequences of the human HAO-1 target sites.

[0217] SEQ ID NO:6954 shows the nucleotide sequence of an sgRNA engineered to function with the MG29-1 nuclease to target cynomolgus monkey HAO-1.

[0218] SEQ ID NOs: 5836-5845 and 6955 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target mouse HAO-1.

[0219] APO-A1 targeting

[0220] SEQ ID NOs:5847-5860 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target mouse APO-A1.

[0221] SEQ ID NOs: 5861-5874 show the DNA sequences of the APO-A1 target sites.

[0222] Mouse ANGPTL3 targeting

[0223] SEQ ID NOs:5875-5952 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target mouse ANGPTL3.

[0224] SEQ ID NOs:5953-6030 show the DNA sequences of mouse ANGPTL3 target sites.

[0225] MG29-1 Human GPR146 Targeting

[0226] SEQ ID NOs:6060-6068 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human GPR146.

[0227] SEQ ID NOs: 6069-6077 show the DNA sequences of human GPR146 target sites.

[0228] GPR146 targeting in MG29-1 mice

[0229] SEQ ID NOs:6078-6079 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target mouse GPR146.

[0230] SEQ ID NOs:6080-6081 show the DNA sequences of the mouse GPR146 target sites.

[0231] Human ANGPTL3 targeting

[0232] SEQ ID NOs:6082-6177 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 nuclease to target human ANGPTL3.

[0233] SEQ ID NOs:6178-6273 show the DNA sequences of human ANGPTL3 target sites.

[0234] Human VCP targeting

[0235] SEQ ID NOs:6551-6556 show the nucleotide sequences of sgRNAs engineered to function with MG29-1 nuclease to target human VCP.

[0236] SEQ ID NOs:6557-6562 show the DNA sequences of the human VCP target sites. DETAILED DESCRIPTION

[0237] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Various changes, modifications and substitutions may be envisioned by 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.

[0238] Unless otherwise indicated, the practice of certain methods disclosed herein employs techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); the book series Current Protocols in Molecular Biology (FM Ausubel et al., eds.); the book series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR 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 (RI Freshney, ed., (2010)).

[0239] 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. In addition, where the terms "including," "include," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0240] 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 will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one or more than one standard deviation, as practiced in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0241] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Envisioned nucleotides include naturally occurring nucleotides and synthetic nucleotides. Nucleotide is a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or its derivatives. Such derivatives include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that impart nuclease resistance to nucleic acid molecules containing it. As used herein, the term nucleotide encompasses dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Illustrative examples of ddNTP include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddTTP. Nucleotide can be unlabeled or detectably labeled, such as using a part or quantum dot comprising an optically detectable portion (e.g., fluorophore). Detectable labeling includes, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. The fluorescent labeling of nucleotides includes, but is 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-carboxyl-X-rhodamine (ROX), 4-(4' dimethylaminophenylazo) benzoic acid (DABCYL), waterfall 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, available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxyribonucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Boehringer Mannheim, Indianapolis, Ind.; 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 Molecular Probes, Eugene, Oreg.; and chromosome-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-FL-5-UTP, available from Molecular Probes, Eugene, Oreg. IPY-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. Exemplary chemically modified nucleotides are biotin-dNTPs.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).

[0242] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides or their analogs, in single-stranded, double-stranded or multi-stranded forms. The contemplated polynucleotides include genes or fragments thereof. Exemplary polynucleotides include, but are not limited to, coding or non-coding regions of DNA, RNA, genes or gene fragments, multiple loci defined according to connection analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, cell-free polynucleotides, nucleic acid probes and primers comprising cell-free DNA (cfDNA) and cell-free RNA (cfRNA). In a polynucleotide, when T is mentioned, T means U (uracil) in RNA and T (thymine) in DNA. Polynucleotides can be exogenous or endogenous to cells and / or present in a cell-free environment. The term polynucleotide encompasses modified polynucleotides (e.g., altered backbones, sugars or nucleobases). If present, modification of the nucleotide structure is imparted before or after assembling the polymer. Non-limiting examples of modification include: 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, locked nucleic acids, glycerol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided glycosides and wyoside. The nucleotide sequence can be interspersed with non-nucleotide components.

[0243] The term "transfection" or "transfected" refers to the introduction of a polynucleotide into a cell by a non-viral or viral-based method. The polynucleotide can be a gene sequence encoding a complete protein or a functional portion thereof. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0244] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues connected by a peptide bond. This term does not indicate the specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally present. The term applies to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interspersed with non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structures (e.g., domains). The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operations, such as conjugation with a labeling component. As used herein, the term "amino acid (amino acid / amino acids)" refers to natural and non-natural amino acids, including but not limited to modified amino acids. Modified amino acids include natural amino acids that have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. The term "amino acid" includes both D-amino acids and L-amino acids.

[0245] As used herein, "non-natural" refers to non-natural nucleic acid or polypeptide sequences. Non-natural refers to non-natural nucleic acid or polypeptide sequences that contain modifications such as mutations, insertions or deletions. The term non-natural encompasses fusion nucleic acids or polypeptides that encode or show activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) of the nucleic acid or polypeptide sequence fused to the non-natural sequence. Non-natural nucleic acids or polypeptide sequences include those non-natural nucleic acids or polypeptide sequences that are connected to naturally occurring nucleic acids or polypeptide sequences (or variants thereof) by genetic engineering to generate chimeric nucleic acids or polypeptide sequences encoding chimeric nucleic acids or polypeptides.

[0246] As used herein, the term "promoter" refers to a regulatory DNA region that controls polynucleotide (e.g., gene) transcription or expression and can be located adjacent to or overlapping a nucleotide or nucleotide region that initiates RNA transcription. A promoter can contain a specific DNA sequence that binds a protein factor (commonly referred to as a transcription factor) that promotes the binding of RNA polymerase to DNA, thereby resulting in gene transcription. Eukaryotic basal promoters typically (although not necessarily) contain a TATA box and / or a CAAT box.

[0247] As used herein, the term "expression" refers to the process of transcribing a nucleic acid sequence or polynucleotide from a DNA template (e.g., transcribed into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, the term expression includes the splicing of mRNA in eukaryotic cells.

[0248] As used herein, "operably connected", "operably connected", "operably connected" or its grammatical equivalent refers to genetic elements, such as the arrangement of promoters, enhancers, polyadenylation sequences, etc., wherein the operation (for example, movement or activation) of the first genetic element has some influence on the second genetic element. The influence on the second genetic element can be but need not be the same type as the operation of the first genetic element. For example, if the movement of the first element causes the activation of the second element, the two genetic elements are operably connected. For example, if the regulatory element helps to start the transcription of the coding sequence, the regulatory element that can contain promoters and / or enhancer sequences is operably connected to the coding region. As long as this functional relationship is maintained, there may be an insertion residue between the regulatory element and the coding region.

[0249] As used herein, "vector" refers to a macromolecule or an associate of macromolecules that contains or associates with a polynucleotide and mediates the 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 include genetic elements (e.g., regulatory elements) that are operably linked to a gene to promote expression of the gene in a target.

[0250] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to components of a vector comprising a combination of nucleic acid sequences or elements (e.g., therapeutic genes, promoters, and terminators) that are expressed together or operably linked for expression. These terms encompass expression cassettes that include a combination of regulatory elements and one or more genes operably linked thereto for expression.

[0251] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence includes its ability to affect expression in a manner attributed to the full-length sequence.

[0252] The terms "engineered", "synthetic" and "artificial" are used interchangeably herein to refer to objects that are modified by human intervention. For example, these terms refer to non-naturally occurring polynucleotides or polypeptides. Engineered peptides have, but do not require, low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) with naturally occurring human proteins. For example, the VPR and VP64 domains are synthetic transactivation domains. Non-limiting examples include the following: nucleic acids are modified by changing their sequence to a sequence that does not exist in nature; nucleic acids are modified by connecting them to nucleic acids that are not associated with them in nature, so that the connection product has a function that does not exist in the original nucleic acid; engineered nucleic acids are synthesized in vitro with sequences that do not exist in nature; proteins are modified by changing the amino acid sequence of proteins to sequences that do not exist in nature; engineered proteins obtain new functions or properties. An "engineered" system comprises at least one engineered component.

[0253] As used herein, the term "Cas12a" refers to a family of Cas endonucleases that belong to class 2, type VA Cas endonucleases and (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.

[0254] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize with a target nucleic acid and thereby guide the relevant nuclease to the target nucleic acid. A guide nucleic acid is, but is not limited to, RNA (guide RNA or gRNA), DNA, or a mixture of RNA and DNA. Guide nucleic acids include crRNA or tracrRNA or a combination of the two. The term guide nucleic acid covers engineered guide nucleic acids and programmable guide nucleic acids to specifically bind to a target nucleic acid. A portion of a target nucleic acid may be complementary to a portion of a guide nucleic acid. The chain of a double-stranded target polynucleotide that is complementary to and hybridized with a guide nucleic acid is a complementary chain. The chain of a double-stranded target polynucleotide that is complementary to a complementary chain and is therefore not complementary to a guide nucleic acid is referred to as a non-complementary chain. A guide nucleic acid with a polynucleotide chain is a "single guide nucleic acid". A guide nucleic acid with two polynucleotide chains is a "double guide nucleic acid". If not otherwise specified, the term "guide nucleic acid" is inclusive and refers to both a single guide nucleic acid and a double guide nucleic acid. A guide nucleic acid may include a segment referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence" or a "spacer". The nucleic acid targeting segment may include sub-segments, which are referred to as "protein binding segments" or "protein binding sequences" or "Cas protein binding segments."

[0255] As used herein, the terms "gene editing" and "genome editing" can be used interchangeably. Gene editing or genome editing means changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0256] As used herein, the term "complex" refers to the connection of at least two components. The two components can each retain the properties / activity they had before forming the complex or acquire properties as a result of forming the complex. Connection includes, but is not limited to, covalent bonding, non-covalent bonding (i.e., hydrogen bonding, ionic interactions, van der Waals interactions, and hydrophobic bonds), use of linkers, fusion, or any other suitable method. The components of the contemplated complex include polynucleotides, polypeptides, or combinations thereof. For example, the complex comprises an endonuclease and a guide polynucleotide.

[0257] The terms "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refer to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example: BLASTP for polypeptide sequences longer than 30 residues using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix with gap penalties set to existence of 11, extension of 1, and adjustment using a conditional composition scoring matrix; BLASTP for sequences less than 30 residues using parameters of a wordlength (W) of 2, an expectation (E) of 1000000, and a PAM30 scoring matrix with gap penalties set to gap open of 9 and extension of gap of 1 (these are the default parameters for BLASTP in the BLAST suite, available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using the Smith-Waterman homology search algorithm parameters of matches of 2, mismatches of -1, and gaps of -1; MUSCLE using default parameters; MAFFT using parameters of retree of 2 and maximum iterations of 1000; Novafold using default parameters; HMMER hmmalign using default parameters.

[0258] In the context of two or more nucleic acid or polypeptide sequences, the term "optimally aligned" generally refers to two (e.g., pairwise) or more (e.g., in a multiple sequence alignment) sequences that have been aligned for maximum correspondence of amino acid residues or nucleotides, for example, as determined by the alignment that produces the highest or "optimizing" percent identity score.

[0259] The present disclosure includes variants of any enzyme in the enzymes described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be carried out in the amino acid sequence of a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be completed by replacing each other with amino acids with similar hydrophobicity, polarity, and R chain length. Additionally or alternatively, by comparing the alignment sequences of homologous proteins from different species, conservative substitutions can be identified by positioning the amino acid residues that mutate between species (e.g., non-conservative residues) without changing the basic function of the encoded protein. Such conservatively substituted variants can include any endonuclease protein sequence in the endonuclease protein sequence described herein (e.g., 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 endonucleases described herein, or Any other family nuclease in the nucleic acid family (of any other family nuclease in the nucleic acid family) has a variant with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions so that the activity of one or more key active site residues of the nucleic acid endonuclease or the guide RNA binding residue is not destroyed. In some embodiments, the functional variants of any protein in the proteins described herein lack substitutions of at least one conservative residue or functional residue. In some embodiments, the functional variants of any protein in the proteins described herein lack substitutions of all conservative residues or functional residues.

[0260] The present disclosure also includes variants of any of the enzymes described herein that replace one or more catalytic residues to reduce or eliminate the activity of the enzyme (e.g., variants with reduced activity). In some embodiments, variants with reduced activity of proteins described herein include disruptive substitutions of at least one, at least two, or all three catalytic residues.

[0261] Conservative substitution tables providing functionally similar amino acids are available from various references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd ed. (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:

[0262] 1) Alanine (A), glycine (G);

[0263] 2) Aspartic acid (D), glutamic acid (E);

[0264] 3) Asparagine (N), glutamine (Q);

[0265] 4) Arginine (R), Lysine (K);

[0266] 5) Isoleucine (I), leucine (L), methionine (M), valine (V);

[0267] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0268] 7) serine (S), threonine (T); and

[0269] 8) Cysteine ​​(C), Methionine (M)

[0270] Overview

[0271] The discovery of new Cas enzymes with unique functions and structures offers the possibility to further gene editing technologies with improved speed, specificity, functionality, and ease of use. Relative to the predicted prevalence of clustered regularly interspaced short palindromic repeat (CRISPR) systems in microorganisms and the sheer diversity of microbial species, relatively few functionally characterized CRISPR / Cas enzymes exist in the literature. This is in part because a large number of microbial species may not be easily cultured under laboratory conditions. Metagenomic sequencing of natural environmental niches containing a large number of microbial species may offer the potential to substantially increase the number of new CRISPR / Cas systems characterized and accelerate the discovery of new oligonucleotide editing functions. A recent example of the fruitfulness of this approach was demonstrated by the discovery of the CasX / CasY CRISPR system in 2016 through metagenomic analysis of natural microbial communities.

[0272] The CRISPR / Cas system is an RNA-guided nuclease complex that acts as an adaptive immune system in microorganisms. In its natural environment, the CRISPR / Cas system occurs in a CRISPR (clustered regularly interspaced short palindromic repeats) operator or locus, and the system consists of two parts: (i) a short repeat array (30-40bp) separated by a short spacer sequence, which encodes an RNA-based targeting element; and (ii) an ORF encoding a Cas nuclease. The efficient nuclease targeting of a specific target nucleic acid sequence generally requires the following two: (i) complementary hybridization between the first 6-8 nucleic acids of the target nucleic acid and the crRNA guide; and (ii) according to a specific Cas nuclease, a protospacer adjacent motif (PAM) sequence (PAM is generally a sequence not often represented in the host genome) exists in a certain vicinity of the target nucleic acid sequence. According to the exact function and organization of the system, the CRISPR-Cas system is generally divided into 2 categories, 5 types, and 16 subtypes based on shared functional properties and evolutionary similarities.

[0273] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include type I, III, and IV Cas nucleases. Class 2 CRISPR-Cas systems typically have single-polypeptide multi-domain nuclease effectors and include type II, V, and VI Cas nucleases.

[0274] Type II CRISPR-Cas systems are considered to be the simplest in terms of components. In type II CRISPR-Cas systems, the CRISPR array is processed into mature crRNA without the presence of special endonuclease subunits, but requires a small trans-encoded crRNA (tracrRNA), whose region is complementary to the array repeat sequence; tracrRNA interacts with its corresponding effector nuclease (e.g., Cas9) and repeat sequence to form a precursor dsRNA structure, which is cut by endogenous RNase III, thereby generating a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are identified as DNA nucleases. Type 2 effectors generally show a structure comprising a RuvC-like endonuclease domain, which is folded using RNase H, wherein the folding of the RuvC-like nuclease domain is inserted with an unrelated HNH nuclease domain. The RuvC-like domain is responsible for the cutting of the target (e.g., crRNA complementary) DNA chain, and the HNH domain is responsible for the cutting of the replaced DNA chain.

[0275] The V-type CRISPR-Cas system is characterized by a nuclease effector (e.g., Cas12) structure similar to the structure of the type II effector containing a RuvC-like domain. Similar to type II, most (but not all) V-type CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA. However, unlike the type II system that requires RNase III to cut pre-crRNA into multiple crRNAs. The V-type system is able to use the effector nuclease itself to cut the crRNA precursor. Like the type II CRISPR-Cas system, the V-type CRISPR-Cas system is again identified as a DNA nuclease. Unlike the type II CRISPR-Cas system, some V-type enzymes (e.g., Cas12a) appear to have a strong single-stranded nonspecific deoxyribonuclease activity activated by the first crRNA-directed cutting of the double-stranded target sequence.

[0276] The CRISPR-Cas system has become the gene editing technology of choice in recent years due to its targeting and ease of use. The most commonly used systems are 2 types of SpCas9, type II, and 2 types of Cas12a, type VA. In particular, the type VA system is becoming more widely used because of its reported specificity in cells that is higher than other nucleases, with fewer or no off-target effects. The VA system also has the advantage that the guide RNA is small (42-44 nucleotides, compared to approximately 100nt for SpCas9) and is processed by the nuclease itself after transcription from the CRISPR array, simplifying multiple applications of multi-gene editing. In addition, the VA system has staggered cleavage sites, which may facilitate directed repair pathways such as microhomology-dependent targeted integration (MITI).

[0277] The most commonly used VA-type enzymes require a 5' protospacer adjacent motif (PAM) next to the selected target site: 5'-TTTV-3' for Lachnospiraceae bacteria ND2006 LbCas12a and Acidaminococcus sp. AsCas12a; and 5'-TTV-3' for Francisella novicida FnCas12a. Recent exploration of orthologs has revealed proteins with less restrictive PAM sequences that are also active in mammalian cell culture, e.g., YTV, YYN, or TTN. However, these enzymes do not fully cover the VA biodiversity and targeting and may not represent all possible activities and PAM sequence requirements. Here, thousands of genomic fragments were identified from a large metagenome of VA-type nucleases. The diversity of identified VA enzymes may have been expanded, and improved systems may have been developed to become highly targeted, compact, and precise gene editors.

[0278] MG enzyme

[0279] In certain embodiments, endonucleases (eg, type V endonucleases) are described herein.

[0280] 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, 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 a MG61 nuclease described herein (e.g., SEQ ID NOs: 1164-1469). In some embodiments, the endonuclease is a MG62 nuclease described herein (e.g., SEQ ID NOs: 1470-1472). In some embodiments, the endonuclease is a MG70 nuclease described herein (e.g., SEQ ID NOs: 1473-1514). In some embodiments, the endonuclease is a MG75 nuclease described herein (e.g., SEQ ID NOs: 1515-1710). In some embodiments, the endonuclease is a MG77 nuclease described herein (e.g., SEQ ID NOs: 1711-1712). In some embodiments, the endonuclease is a MG78 nuclease described herein (e.g., SEQ ID NOs: 1713-1717). In some embodiments, the endonuclease is a 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 NO: 1724-2654). In some embodiments, the endonuclease is an MG82 nuclease described herein (e.g., SEQ ID NO: 2655-2657). In some embodiments, the endonuclease is an MG83 nuclease described herein (e.g., SEQ ID NO: 2658-2659). In some embodiments, the endonuclease is an MG84 nuclease described herein (e.g., SEQ ID NO: 2660-2677). In some embodiments, the endonuclease is an MG85 nuclease described herein (e.g., SEQ ID NO: 2678-2680). In some embodiments, the endonuclease is an MG90 nuclease described herein (e.g., SEQ ID NO: 2681-2809). In some embodiments, the endonuclease is an MG91 nuclease described herein (eg, SEQ ID NOs: 2810-3470 and 6274-6281).

[0281] In some embodiments, the length of the endonuclease described herein is about 1000-1100 amino acids. In some embodiments, the length of the endonuclease described herein is less than about 400 amino acids. In some embodiments, the length of the endonuclease described herein is about 500-700 amino acids. In some embodiments, the endonuclease described herein comprises RuvC and HTH DNA binding domains. In some embodiments, the endonuclease comprises RuvCI, II or III domains. 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 endonuclease further comprises a WED II domain. In some embodiments, the endonuclease further comprises a zinc finger domain. In some embodiments, the endonuclease described herein does not require tracrRNA.

[0282] In some embodiments, the endonuclease is a Cas endonuclease. In some embodiments, the endonuclease is a Class 2 V-type Cas endonuclease. In some embodiments, the endonuclease is a Class 2 VA-type Cas endonuclease. In some embodiments, the endonuclease is not a Cpf1 or Cms1 endonuclease. In some embodiments, the endonuclease further comprises a zinc finger domain.

[0283] 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 is 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% identical 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.

[0284] 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 is 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% identical 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.

[0285] In some embodiments, the endonuclease is an MG91 endonuclease (e.g., SEQ ID NOs: 6274-6281). In some embodiments, the endonuclease comprises a sequence having at least about 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 that is at least about 98% identical to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6274-6281. In some embodiments, the endonuclease comprises a sequence that is 100% identical to any one of SEQ ID NOs: 6274-6281.

[0286] In some embodiments, the endonuclease is an MG29 endonuclease (eg, 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 is 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% identical 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 that is 100% identical to any one of SEQ ID NOs: 6340-6550 and 6563-6565.

[0287] 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.

[0288] 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 with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 85% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 90% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 95% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 96% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 97% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence having at least about 98% identity with TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence that is at least about 99% identical to TtTYn, GnYYn, or wCCC. In some embodiments, the PAM sequence comprises a sequence that is 100% identical to TtTYn, GnYYn, or wCCC.

[0289] 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 that is 100% identical to tnTYn, GnGYCn, TTTY, Cc, Gnkynn, ttTYnAA, nnnCn, yYt, yYy, TtGc, tngn, gngY, mCm, or ryCC.

[0290] 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 that is at least about 75% identical 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 that is at least about 80% identical 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 that is at least about 85% identical 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 that is at least about 90% identical 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 that is at least about 95% identical 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 that is at least about 96% identical 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 that is at least about 97% identical 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 that is at least about 98% identical 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 that is at least about 99% identical 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 that is 100% identical to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, and 6281.

[0291] In some embodiments, endonucleases are found by metagenomic sequencing. In some embodiments, the metagenomic sequencing is performed on a sample. In some embodiments, samples are collected from a variety of environments. Such environments can be human microbial groups, animal microbial groups, high temperature environments, low temperature environments. Such environments can include sediments.

[0292] In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) near the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises the sequence of any one of SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises the sequence of 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%, at least about 99% identity with 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 NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 90% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 91% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 92% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 93% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 94% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 95% identity to SEQ ID NOs: 3938-3953. In some embodiments, the NLS comprises a sequence having at least about 96% identity to SEQ ID NOs: 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.

[0293] Table 1: Example NLS sequences that can be used with Cas effectors according to the present disclosure.

[0294]

[0295]

[0296] Guide polynucleotide

[0297] In certain embodiments, disclosed herein is an engineered endonuclease system comprising: (a) an endonuclease disclosed herein; and (b) an engineered guide polynucleotide, e.g., a guide RNA (gRNA), a single gRNA, or a dual guide RNA. In a polynucleotide, when T is mentioned, T means U (uracil) in RNA and T (thymine) in DNA.

[0298] 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 with a target nucleic acid sequence. In some embodiments, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0299] 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 pig, cattle, goat, sheep, 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.

[0300] In some embodiments, the guide polynucleotide (e.g., gRNA) comprises NO: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 have at least 80% sequence identity to the first 19 nucleotides or non-degenerate nucleotides of any one of. In some embodiments, the guide polynucleotide comprises a sequence having at least 80% sequence identity to SEQ ID NO: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 6 The first 19 nucleotides or non-degenerate nucleotides of any one of 567-6581 have a sequence that is 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% identical. In some embodiments, the guide polynucleotide comprises a sequence that is 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.

[0301] In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is at least about 85% identical 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 that is at least about 90% identical 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 that is at least about 95% identical 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 that is at least about 96% identical 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 that is at least about 97% identical 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 that is at least about 98% identical 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 that is at least about 99% identical 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 that is 100% identical 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.

[0302] 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 that is at least about 97% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581.

[0303] 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 that is 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% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4428-4465 and 5685.

[0304] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4428-4465 and 5685. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4428-4465 and 5685.

[0305] 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 that has 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 that has 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 that has 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 that has 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 that has 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 that has 100% identity to any one of SEQ ID NOs: 4466-4503 and 5686.

[0306] 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 that is 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% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4504-4520.

[0307] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4504-4520. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4504-4520.

[0308] 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 that is at least about 96% identical to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4521-4537. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 4521-4537.

[0309] 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 that is 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% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4538-4568.

[0310] 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4538-4568. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4538-4568.

[0311] 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 that is at least about 96% identical to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4569-4599. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 4569-4599.

[0312] 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 that is 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% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4600-4675.

[0313] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4600-4675. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4600-4675.

[0314] 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 that is at least about 96% identical to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4676-4751. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 4676-4751.

[0315] 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 that is 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% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4752-4836.

[0316] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4752-4836. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4752-4836.

[0317] 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 that is at least about 96% identical to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4837-4921. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 4837-4921.

[0318] 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 that is 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% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 4922-4945.

[0319] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4922-4945. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4922-4945.

[0320] 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 with 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 with 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 with 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 with 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 with any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4946-4969. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 4946-4969.

[0321] 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 that is 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% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is 100% identical to any one of SEQ ID NOs: 4970-5012.

[0322] 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 4970-5012. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 4970-5012.

[0323] 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 that is at least about 95% identical to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5013-5055. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5013-5055.

[0324] 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 that is 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% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is at least about 98% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683.In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683.

[0325] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 90% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5056-5125, 5681, and 5683.

[0326] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within a 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 that has 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 that has 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 that has 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 that has 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 that has 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 that is at least about 99% identical to any one of SEQ ID NOs: 5126-5195, 5682, and 5684. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5126-5195, 5682, and 5684.

[0327] 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 that is 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% identical 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 that is at least about 99% identical 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 that is 100% identical to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660.

[0328] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is complementary to a sequence having 100% identity to any one of SEQ ID NOs: 5196-5210, 5226-5246, and 5642-5660.

[0329] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within a 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, the 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, the 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, the 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 that is at least about 97% identical 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 that is at least about 98% identical 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 that is at least about 99% identical 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 that is 100% identical to any one of SEQ ID NOs: 5211-5225, 5247-5267, and 5661-5679.

[0330] 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 that is 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% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 5268-5366.

[0331] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5268-5366. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5268-5366.

[0332] 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 that is at least about 96% identical to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5367-5465. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5367-5465.

[0333] 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 that is 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% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 5466-5473.

[0334] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5466-5473. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5466-5473.

[0335] 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 with 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 with 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 with 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 with 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 with any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5474-5481. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5474-5481.

[0336] 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 that is 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% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 5482-5561.

[0337] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5482-5561. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5482-5561.

[0338] 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, the 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, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5562-5641. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5562-5641.

[0339] 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 that is 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% identical 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 that is at least about 95% identical 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 that is at least about 96% identical 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 that is 100% identical to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.

[0340] 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 that is 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% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is at least about 99% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 6909-6930 and 6953.

[0341] 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 that is 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% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955.

[0342] 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: 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 hybridizes or targets a sequence that is 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 that is 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 that is 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 that is complementary to a sequence that is at least about 95% identical 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 that is complementary to a sequence that is at least about 96% identical 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 that is complementary to a sequence that is at least about 97% identical 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 that is complementary to a sequence that is at least about 98% identical 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 that is complementary to a sequence that is at least about 99% identical 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 that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5788-5829, 5831-5834, 5836-5845, 6909-6930, and 6953-6955.

[0343] 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 that is complementary to a sequence that is at least about 90% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6909-6930 and 6953. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 6909-6930 and 6953.

[0344] 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 that is complementary to a sequence that is at least about 95% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to SEQ ID NO: 6954 or SEQ ID NO: 6955.

[0345] 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, the 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, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6931-6952. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 6931-6952.

[0346] 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 that is 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% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 5847-5860.

[0347] 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5847-5860. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 5847-5860.

[0348] 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 that is at least about 95% identical to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 5861-5874. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 5861-5874.

[0349] 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 that is 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% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 6082-6177.

[0350] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within an 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6082-6177. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 6082-6177.

[0351] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within an 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 that is at least about 96% identical to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6178-6273. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 6178-6273.

[0352] 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 that is 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% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is 100% identical to any one of SEQ ID NOs: 6060-6068.

[0353] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6060-6068. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 6060-6068.

[0354] 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 that is at least about 95% identical to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6069-6077. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 6069-6077.

[0355] 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 that is 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% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical 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 that is 100% identical to any one of SEQ ID NOs: 6078-6079.

[0356] 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6078-6079. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 6078-6079.

[0357] 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, the guide polynucleotide hybridizes or targets a sequence that has at least about 95% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that has at least about 96% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that has at least about 97% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that has at least about 98% identity to any one of SEQ ID NOs: 6080-6081. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that has 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 that has 100% identity to any one of SEQ ID NOs: 6080-6081.

[0358] 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 that is 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% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 80% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 85% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 90% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide is encoded by a sequence that is 100% identical to any one of SEQ ID NOs: 6551-6556.

[0359] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within a 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 that is complementary to a sequence that is at least about 95% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6551-6556. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to a sequence that is 100% identical to any one of SEQ ID NOs: 6551-6556.

[0360] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within a 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, the 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, the guide polynucleotide hybridizes or targets a sequence that is at least about 96% identical to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 97% identical to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 98% identical to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is at least about 99% identical to any one of SEQ ID NOs: 6557-6562. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is 100% identical to any one of SEQ ID NOs: 6557-6562.

[0361] In some embodiments, the guide polynucleotide is configured to form a complex with an endonuclease. In some embodiments, the guide polynucleotide binds to an endonuclease to form a complex. In some embodiments, the guide polynucleotide binds to an endonuclease (e.g., non-covalently via electrostatic interactions or hydrogen bonds) to form a complex. In some embodiments, the guide polynucleotide is fused to an endonuclease to form a complex.

[0362] In some embodiments, the 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.

[0363] 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 pig, cow, goat, sheep, rodent, rat, mouse, non-human primate, or human cell.

[0364] In some embodiments, the guide polynucleotide (e.g., guide RNA) includes various structural elements, including but not limited to: a spacer sequence, crRNA, and optional tracrRNA that binds to a protospacer sequence (target sequence). In some embodiments, the genome editing system includes a CRISPR guide RNA. In some embodiments, the guide RNA includes a crRNA that includes a spacer sequence. In some embodiments, the guide RNA additionally includes tracrRNA or modified tracrRNA.

[0365] 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 that is complementary or substantially complementary to a region of a target sequence. For example, a crRNA is or is considered to be part of a guide polynucleotide, or is contained in a guide polynucleotide, such as a crRNA:tracrRNA chimera.

[0366] In some embodiments, the guide polynucleotide comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide polynucleotide comprises one or more internucleoside linkers modified by natural phosphodiester. In some embodiments, all internucleoside linkers of the guide polynucleotide or a contiguous nucleotide sequence thereof are modified. For example, in some embodiments, the internucleoside bond comprises sulfur (S), such as a thiophosphate internucleoside bond. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% of 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., thiophosphate internucleoside bonds).

[0367] In some embodiments, the guide polynucleotide comprises a modification to a ribose sugar or a nucleobase. In some embodiments, the guide polynucleotide comprises one or more nucleosides comprising a modified sugar moiety, wherein a modified glucose moiety is a modification of the sugar moiety when compared to the ribose sugar moiety present in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a diradical bridge between the C2 carbon and the C4 carbon on the ribose ring (e.g., locked nucleic acid (LNA)), or an unconnected ribose ring (e.g., UNA) that typically lacks a bond between the C2 carbon and the C3 carbon. In some embodiments, sugar-modified nucleosides comprise bicyclic hexose nucleic acids or tricyclic nucleic acids. In some embodiments, modified nucleosides comprise nucleosides in which the sugar moiety is replaced by a non-sugar moiety, such as a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0368] In some embodiments, the guide polynucleotide comprises one or more modified sugars. In some embodiments, the sugar modification comprises modification by changing the substituent on the ribose ring to a group other than hydrogen or 2'-OH groups naturally present 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, the nucleosides having a modified sugar moiety comprise 2' modified nucleosides, e.g., 2' substituted nucleosides. In some embodiments, 2' sugar modified nucleosides are nucleosides having a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or comprising a 2' linked diradical, and comprise 2' substituted nucleosides and LNA (2'-4' diradical 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 includes 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).

[0369] 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 greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a disaccharide. In some embodiments, the modified sugar comprises 2'-O-methyl. In some embodiments, the modified sugar comprises 2'-fluoro. In some embodiments, the modified sugar comprises 2'-O-methoxyethyl. 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).

[0370] In some embodiments, the guide polynucleotide comprises both an internucleoside linker modification and a nucleoside modification. In some embodiments, the guide polynucleotide comprises greater than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers and greater 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).

[0371] 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 4 bases of the 5' end of the guide polynucleotide or within the last 4 bases of the 3' end of the guide polynucleotide; (ii) a phosphorothioate (PS) bond between at least 2 bases of the first five bases of the 5' end of the guide polynucleotide, or a phosphorothioate bond between at least two bases of the last five bases of the 3' end of the guide polynucleotide; (iii) a phosphorothioate bond within the 3' stem or 5' stem of the guide polynucleotide; (iv) a 2'-O methyl or 2' base modification within the 3' stem or 5' stem of the guide polynucleotide; (v) a 2'-fluoro base modification of at least 7 bases of the spacer region of the guide polynucleotide; and (vi) a phosphorothioate bond 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 5 bases of the 5' end of the guide polynucleotide or within the last 5 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 at the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a phosphorothioate (PS) bond between at least 2 bases of the first five bases of the 5' end of the guide polynucleotide or a phosphorothioate bond between at least two bases of the last five bases of the 3' end of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a phosphorothioate bond within the 3' stem or 5' stem of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-O methyl base modification within the 3' stem or 5' stem of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises a 2'-O methyl base modification of at least 7 bases of the spacer region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises phosphorothioate bonds 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 region or the 5' stem region of the guide polynucleotide. In some embodiments, the guide polynucleotide comprises at least one to at least fourteen 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 one to at least fourteen 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 nucleotides of any one of SEQ ID NOs: 3985-3991, the guide RNA comprising a chemical modification listed in SEQ ID NOs: 3985-3991. In some embodiments, the RNA-guided nuclease is a Cas endonuclease. In some embodiments, the Cas endonuclease is a Class 2 V-type Cas endonuclease. In some embodiments, the 2 types of V-type Cas endonucleases include a RuvC domain comprising a RuvCI subdomain, a RuvCII subdomain, and a RuvCIII subdomain. In some embodiments, the 2 types of V-type Cas endonucleases include an endonuclease with at least 75% sequence identity to any one of SEQ ID NOs: 1-3470, 6272-6281, 6340-6550, and 6563-6565, or a variant thereof. In some embodiments, the 2 types of V-type Cas endonucleases include an endonuclease with at least 75% sequence identity to any one of SEQ ID NOs: 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.

[0372] 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.

[0373] In some embodiments, the guide polynucleotide is 30-250 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 polynucleotide is about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 1 In some embodiments, the present invention relates to a polypeptide having at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the polypeptide has at least one nucleotide sequence and at least one nucleotide sequence. In some embodiments, the polypeptide has at least one nucleotide sequence and at least one nucleotide sequence.

[0374] In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 8 base-paired ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 9 base-paired ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 10 base-paired ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 11 base-paired ribonucleotides. In some embodiments, the guide polynucleotide comprises a hairpin comprising at least 12 base-paired ribonucleotides.

[0375] In some embodiments, the guide polynucleotide comprises a DNA targeting segment. In some embodiments, the DNA targeting segment comprises a nucleotide sequence 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 nucleotide extensions. In some embodiments, the two complementary nucleotide extensions hybridize to form a double-stranded RNA (dsRNA) duplex. In some embodiments, the two complementary nucleotide extensions are covalently linked to each other with an intermediate nucleotide.

[0376] In some embodiments, the DNA targeting segment is positioned 3' to both of the two complementary nucleotide stretches. In some embodiments, the protein binding segment comprises a sequence that is 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% identical to the first 19 nucleotides or non-degenerate nucleotides of SEQ ID NO: 3608.

[0377] 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.

[0378] MG Endonuclease System

[0379] In certain embodiments, described herein are engineered nuclease systems 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.

[0380] 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 70% identity. 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 75% identity.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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 80% identity. 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 85% identity.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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 90% identity. 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 95% identity.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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 96% identity. 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 97% identity.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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 98% identity. 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: 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 engineered guide polynucleotide 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. any one of 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 having a sequence with at least about 99% identity.In some embodiments, the engineered nuclease system comprises an endonuclease comprising 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 engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 1-3470, 6274-6281, 6340-6550, and 6563-6565. Any of 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 has 100% identity. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary 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, or a sequence complementary to any one of SEQ ID NOs: any of 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 having a sequence with at least 90%, 95%, 97%, 98%, or 99% sequence identity.

[0381] 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 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 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 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 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 nuclease 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 nuclease 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 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 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 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 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 nuclease 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 nuclease 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 an endonuclease comprising 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 100% identity to SEQ ID NO: 3612. In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to SEQ ID NO: 3612, or a sequence that has at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3612.

[0382] 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: 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 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 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 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 at least about 90% identical 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 at least about 90% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581.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: 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 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 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 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 at least about 99% identical 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 at least about 99% identical to any one of SEQ ID NOs: 6284-6325 and 6567-6581.In some embodiments, the engineered nuclease system comprises an endonuclease comprising 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 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.

[0383] 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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 with at least a portion of a target nucleic acid sequence within a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 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.

[0384] 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 70% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 75% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 80% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 85% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 90% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 95% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 96% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 97% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 98% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of 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 a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 6954 or 6955. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to SEQ ID NOs: 6954 or 6955 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 6954 or 6955.

[0385] 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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 with at least a portion of a target nucleic acid sequence within a human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising 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.

[0386] 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 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 a mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 6078-6079. 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.

[0387] 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising 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 at least about 99% identical 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 an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence at least about 99% identical 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 an 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.

[0388] 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 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 a VCP gene or within an intron of the VCP gene,

[0389] 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 95% 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 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 96% 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 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 97% 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 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 98% 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 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 a VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 6551-6556. 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, wherein the spacer sequence is configured to hybridize to at least a portion of the target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising 100% identity to any one of SEQ ID NOs: 6551-6556.In some embodiments, the guide polynucleotide hybridizes or targets a sequence that is complementary to any one of SEQ ID NOs: 6551-6556, or a sequence that has at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 6551-6556.

[0390] In some embodiments, the engineered nuclease system further comprises a single-stranded or double-stranded DNA repair template. In some embodiments, the engineered nuclease system further comprises a single-stranded DNA repair template. In some embodiments, the engineered nucleas...

Claims

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; and b) an engineered guide polynucleotide configured to form a complex with the endonuclease and hybridize to a target nucleic acid sequence.

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

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

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

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

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

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

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

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

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

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

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

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

26. 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 nuclease and comprising a spacer sequence configured to hybridize with at least a portion of a target nucleic acid sequence within a HAO-1 gene or within an intron of the HAO-1 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6909-6930 and 6953.

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. 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 nuclease and comprising a spacer sequence, wherein the spacer sequence is configured to hybridize with at least a portion of the target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068.

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. 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 nuclease and comprising a spacer sequence, wherein the spacer sequence is configured to hybridize with at least a portion of the target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079.

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. 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 nuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within an ANGPTL3 gene or within an intron of the ANGPTL3 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177.

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. 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 nuclease and comprising a spacer sequence, wherein the spacer sequence is configured to hybridize with at least a portion of the target nucleic acid sequence within the VCP gene or within an intron of the VCP gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6551-6556.

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

37. An engineered nuclease system according to any one of claims 1 to 35, further comprising a single-stranded or double-stranded DNA repair template, which comprises from 5' to 3': a first homology arm, which comprises a sequence of at least 20 nucleotides located 5' of the target deoxyribonucleic acid sequence; a synthetic DNA sequence of at least 10 nucleotides; and a second homology arm, which comprises a sequence of at least 20 nucleotides located 3' of the target sequence.

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

39. The engineered nuclease system of claim 37 or 38, wherein the first homology arm and the second homology arm are homologous to a genomic sequence of a prokaryote, a bacterium, a fungus or a eukaryote.

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

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

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

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

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

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. A method for modifying a human GPR146 gene, the method comprising contacting the human GPR146 gene with an engineered nuclease system, the 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 nuclease and comprising a spacer sequence, wherein the spacer sequence is configured to hybridize with at least a portion of a target nucleic acid sequence within the human GPR146 gene or within an intron of the human GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6060-6068.

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. A method for modifying a mouse GPR146 gene, the method comprising contacting the mouse GPR146 gene with an engineered nuclease system, the 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 nuclease and comprising a spacer sequence, wherein the spacer sequence is configured to hybridize with at least a portion of the target nucleic acid sequence within the mouse GPR146 gene or within an intron of the mouse GPR146 gene, the engineered guide polynucleotide comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6078-6079.

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. A method of modifying an ANGPTL3 gene, the method 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 nuclease 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 a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 6082-6177.

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

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

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

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

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

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

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

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

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

68. 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 cell, HT1080, HepG2, Huh7, K562, a primary cell, or a derivative thereof.

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

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

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

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

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

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-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA or C12-200.

Images