Engineered and Chimeric Nucleases

Abstract

Disclosed herein are engineered nucleases and nuclease systems, including chimeric nucleases and chimeric nuclease systems. The engineered nucleases and chimeric nucleases disclosed herein include nucleic acid-guided nucleases. Additionally, disclosed herein are methods of producing and using the engineered nucleases.

Description

[Technical Field]

[0001] cross reference

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 319,725, filed March 14, 2022, U.S. Provisional Patent Application No. 63 / 335,542, filed April 27, 2022, U.S. Provisional Patent Application No. 63 / 392,814, filed July 27, 2022, and U.S. Provisional Patent Application No. 63 / 482,294, filed January 30, 2023, each of which is incorporated by reference in its entirety. [Background technology]

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

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on March 14, 2023, is named MTG-010WO_SL.xml and is 4,575,581 bytes in size. Summary of the Invention

[0005] In some aspects, the disclosure provides methods of disrupting the VCP locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the VCP locus, wherein the engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 80% identity to SEQ ID NOs: 739-754 or 763-770, or wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 723-738 or 755-762. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the engineered nucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease having at least 55% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence of 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.In some embodiments, the engineered guide RNA is complementary to or comprises a sequence having 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: 743-745, 749-752, 754, or 769. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 22B comprising a chemical modification listed in Table 22B.

[0006] In some cases, the disclosure provides a method for producing a nucleic acid sequence having (a) at least 55%, 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 SEQ ID NO: 696 or a variant thereof. (b) an N-terminal sequence containing at least a portion of the RuvC-I domain, REC domain, RuvC-II domain, HNH domain, or RuvC-III domain of an endonuclease; and (c) MG3-1, MG3-2, MG3-3, MG3-4, MG3-5, MG3-6, MG3-7, MG3-8, MG3-18, MG3-22, MG3-24, MG3-30, MG3-42, MG3-78, MG3-89, MG3-90, MG3-91, MG3-92, MG3-93, MG3-94, MG3-95, MG3-96, MG3-101, MG3-102, MG3-103, MG3-104, MG3-105, MG3-106, MG3-107, MG3-108, MG3-109, MG3-200, MG3-201, MG3-202, MG3-203, MG3-204, MG3-205, MG3-206, MG3-207, MG3-208, MG3-209, MG3-300, MG3-310, MG3-311, MG3-312, MG3-313, MG3-314, MG3-315, MG3-316, MG3-317, MG3-318, MG3-319, MG3-320, MG3-321, MG3-322, MG3-323, MG3-324, MG3-325, MG3-326, MG3-327, MG3-3 G3-103, MG3-104, MG150-1, MG150-2, MG150-3, MG150-4, MG150-5, MG150-6, MG150-7, MG150-8, MG150-9, MG150-10, MG15-1, MG15-54, MG15-66, MG15-94, MG15-115, MG15-135, MG15-146, MG15-164, MG15-166, MG15-171, MG15-172, MG15-174, MG15-177, MG15-184, MG15-187, MG15-191, MG1 at least 55%, 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% for MG15-193, MG15-195, MG15-217, MG15-218, or MG15-219, or variants thereof;or a C-terminal sequence comprising a WED or PI domain of an endonuclease having at least 99% sequence identity thereto, wherein the N-terminal sequence and the C-terminal sequence do not naturally occur together in the same reading frame. In some embodiments, the N-terminal sequence and the C-terminal sequence are derived from different organisms. In some embodiments, the fusion endonuclease comprises a sequence having at least 55%, 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: 771-862, or a variant thereof. In some embodiments, the fusion endonuclease is configured to be selective for a PAM that is not nnRGGnT. In some embodiments, the fusion endonuclease is configured to be selective for a PAM comprising any one of SEQ ID NOs: 865-919. In some embodiments, the fusion endonuclease comprises a WED domain from an endonuclease having at least 55% sequence identity to MG3-8 and a PAM comprising any one of MG3-1, MG3-2, MG3-3, MG3-4, MG3-5, MG3-6, MG3-7, MG3-8, MG3-18, MG3-22, MG3-24, MG3-30, MG3-42, MG3-78, MG3-89, MG3-90, MG3-91, MG3-92, MG3-93, MG3-94, MG3-95, MG3-96, MG3 -101, MG3-103, MG3-104, MG150-1, MG150-2, MG150-3, MG150-4, MG150-5, MG150-6, MG150-7, MG150-8, MG150-9, MG150-10, MG15-1, MG 15-54, MG15-66, MG15-94, MG15-115, MG15-135, MG15-146, MG15-164, MG15-166, MG15-171, MG15-172, MG15-174, MG15-177, MG15-184,and a PI domain from an endonuclease having at least 55% sequence identity to at least one of MG15-187, MG15-191, MG15-193, MG15-195, MG15-217, MG15-218, or MG15-219, or a variant thereof.

[0007] In some aspects, the present disclosure provides endonucleases comprising an engineered amino acid sequence having at least 55%, 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:771-862, or a variant thereof. In some embodiments, the endonuclease comprises an engineered amino acid sequence having at least 55%, 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: 809-823, 827, 829-830, 832, 834, or 838-839.

[0008] In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) any of the endonucleases described herein; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the guide ribonucleic acid structure comprising a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence, wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the endonuclease comprises a sequence having at least 55%, 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: 771-862, or a variant thereof.In some embodiments, the guide ribonucleic acid sequence comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0009] In some aspects, the disclosure provides methods of disrupting a TRAC locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRAC locus, and wherein the engineered guide RNA hybridizes to a region of the TRAC locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138 The guide RNA is configured to hybridize to, comprises, or is engineered to hybridize to a targeting sequence having 6%, at least 97%, at least 98%, or at least 99% sequence identity, including a sequence having 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: 922-924, 972-991, 1088-1183, or 1280-1320.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein or comprises a sequence having at least 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 SEQ ID NO:771. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0010] In some aspects, the disclosure provides methods of disrupting the AAVS1 locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the AAVS1 locus, and wherein the engineered guide RNA hybridizes to a region of the AAVS1 locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, A guide RNA configured to hybridize to, comprise or engineered to a targeting sequence having 6%, at least 97%, at least 98%, or at least 99% sequence identity includes a sequence having 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: 928-949 or 1012-1049.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein or comprises a sequence having at least 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 SEQ ID NO:771. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0011] In some aspects, the disclosure provides a method of disrupting the PCSK9 locus in a cell, comprising: and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the PCSK9 locus, wherein the engineered guide RNA is configured to hybridize to or comprises a targeting sequence having 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 SEQ ID NOs: 1377-1391, or wherein the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 1362-1376. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease having at least 55%, 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 SEQ ID NO: 10 or a variant thereof.In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0012] In some aspects, the disclosure provides methods of disrupting the ANGPTL3 locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the ANGPTL3 locus, and wherein the engineered guide RNA hybridizes to a region of the ANGPTL3 locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 1109%, at least 1111%, at least 1120%, at least 1121%, at least 1122, at least 1123, at least 1124, at least 1125, at least 1130, at least 1131, at least 1132, at least 1133, at least 1134, at least 1140, at least 1141, at least 1142, at least 1143 The guide RNA may be configured to hybridize to, comprise, or be engineered to hybridize to a targeting sequence having 96%, at least 97%, at least 98%, or at least 99% sequence identity, including a sequence having 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: 1392-1489. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease having at least 55%, 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 SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0013] In some aspects, the disclosure provides methods of disrupting the GPR146 locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the GPR146 locus, and wherein the engineered guide RNA hybridizes to a region of the GPR146 locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least The guide RNA may be configured to hybridize to, comprise, or be engineered to hybridize to a targeting sequence having 96%, at least 97%, at least 98%, or at least 99% sequence identity, including a sequence having 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: 1588-1656. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein.In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0014] In some aspects, the disclosure provides methods of disrupting the APOA1 locus in a cell, the method comprising introducing into the cell (a) a Class 2, Type II Cas endonuclease; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the APOA1 locus, and wherein the engineered guide RNA hybridizes to a region of the APOA1 locus that is 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%, or at least 96% of SEQ ID NOs: 1745-1763 or 1775-1785. The engineered guide RNA is configured to hybridize to, or comprises, a targeting sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOS: 1726-1744 or 1764-1774. The engineered guide RNA comprises a sequence with 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: 1726-1744 or 1764-1774. In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease described herein.In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722 or SEQ ID NO:863.

[0015] In some aspects, the present disclosure provides lipid nanoparticles comprising (a) any of the endonucleases described herein, (b) any of the engineered guide RNAs described herein, (c) a cationic lipid, (d) a sterol, (e) a neutral lipid, and (f) 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 any of the cationic lipids shown in Figure 45.

[0016] In some aspects, the disclosure provides a method for detecting a target polypeptide comprising: (a) an N-terminal sequence comprising at least a portion of the RuvC domain, the REC domain, or the HNH domain of an endonuclease 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 SEQ ID NO: 696 or a variant thereof; and (b) a polypeptide ... and a C-terminal sequence comprising the WED, TOPO, or CTD domain of an endonuclease 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 one or a variant thereof, wherein the N-terminal and C-terminal sequences do not naturally occur together in the same reading frame. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the endonuclease is a Class 2, Type V Cas endonuclease. In some embodiments, the N-terminal sequence and the C-terminal sequence are from different organisms. In some embodiments, the N-terminal sequence further comprises a RuvC-I, BH, or RuvC-II domain. In some embodiments, the C-terminal sequence further comprises a PAM-interacting domain.In some embodiments, the fusion endonuclease 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: 1-27 or 108. In some embodiments, the fusion endonuclease is configured to bind to a PAM that is not nnRGGnT.

[0017] In some aspects, the present disclosure provides endonucleases comprising an engineered amino acid 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: 1-27 or 108, or a variant thereof.

[0018] In some aspects, the present disclosure provides endonucleases comprising an engineered amino acid 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: 109-110, or a variant thereof.

[0019] In some aspects, the disclosure provides nucleic acids comprising a sequence encoding any of the endonucleases, fusion endonucleases, or Cas enzymes described herein. In some aspects, the sequence is codon-optimized for expression in a host cell. In some embodiments, the host cell is prokaryotic, eukaryotic, mammalian, or human.

[0020] In some aspects, the disclosure provides vectors comprising any of the nucleic acid sequences described herein.

[0021] In some aspects, the disclosure provides a host cell comprising any of the vectors, systems, or nucleic acids described herein. In some embodiments, the host cell is prokaryotic, eukaryotic, mammalian, or human.

[0022] In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) any of the nucleases, Cas enzymes, or fusion endonucleases described herein; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the guide ribonucleic acid structure comprising a guide ribonucleic acid configured to hybridize to a target deoxyribonucleic acid sequence, wherein the guide ribonucleic acid sequence is configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to a SpyCas9 endonuclease. In some embodiments, the system comprises a Mg 2+In some embodiments, the endonuclease 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: 8-12, 26-27, or 108, or a variant thereof. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having 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% identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 33, 34, 44, 45, 78, 84, or 87.

[0023] In some aspects, the disclosure provides a Class 2, Type II antibody having (a) 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 the RuvC or HNH domain of any one of SEQ ID NOs: 1-27, 108, or 109-110, or a variant thereof. Provided are engineered nucleases comprising: (a) a Cas enzyme RuvC or HNH domain; and (b) a Class 2, Type II Cas enzyme PAM-interacting (PI) domain 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 the PAM-interacting (PI) domain of any one of SEQ ID NOS: 1-27, 108, or 109-110, or variants thereof. In some embodiments, (a) and (b) do not occur together in nature. In some embodiments, the Class 2, Type II Cas enzyme is derived from an uncultured microorganism. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease.In some embodiments, the engineered nuclease 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: 1-27, or a variant thereof.

[0024] In some aspects, the present disclosure provides an engineered nuclease system comprising: (a) any of the endonucleases described herein; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the guide ribonucleic acid structure being configured to hybridize to a target deoxyribonucleic acid sequence and comprising a guide ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid further comprises a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having 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 the non-degenerate nucleotides of any one of SEQ ID NOS: 28-32 or 33-44, or variants thereof. In some embodiments, the system further comprises a PAM sequence compatible with a nuclease adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located 3' of the target deoxyribonucleic acid sequence. In some embodiments, the PAM sequence is located 5' of the target deoxyribonucleic acid sequence.

[0025] In some aspects, the disclosure provides a method of targeting the albumin gene, comprising introducing any of the systems described herein into a cell, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence having 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: 67-86. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0026] In some aspects, the disclosure provides methods of targeting the HAO1 gene or locus, comprising introducing into a cell any of the systems described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence having 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:611-633. In some embodiments, the guide ribonucleic acid sequence is configured to hybridize to a sequence having 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: 615, 618, 620, 624, or 626. In some embodiments, the guide ribonucleic acid comprises a sequence having 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: 605-610 or 1789-1865.In some embodiments, the guide ribonucleic acid has 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% identity to any one of SEQ ID NOs: 1812-1824, 1835, 1849, 1858, or 1861. or a sequence having 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% identity to the targeting sequence of any one of SEQ ID NOs: 1812-1824, 1835, 1849, 1858, or 1861. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0027] In some embodiments, the disclosure provides methods of disrupting the HAO-1 locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the HAO-1 locus, and wherein the engineered guide RNA is configured to hybridize to or comprises a targeting sequence having 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 SEQ ID NOs:611-633. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease 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% identity to SEQ ID NO: 10 or a variant thereof.In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722. In some embodiments, the engineered guide RNA has 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: 618, 620, 624, or 626. or a sequence having 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 the targeting sequence of any one of SEQ ID NOs: 618, 620, 624, or 626. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Tables 9A-9E, Table 12, Table 18, Table 20, or Table 23. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Tables 9A-9E comprising a chemical modification listed in Tables 9A-9E. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 12, comprising a chemical modification listed in Table 12.In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 18, comprising a chemical modification listed in Table 18. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 20, comprising a chemical modification listed in Table 20. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 23, comprising a chemical modification listed in Table 23. In some embodiments, the cell is a mammalian cell. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0028] In some aspects, the disclosure provides methods of disrupting a TRAC locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRAC locus, and wherein the engineered guide RNA hybridizes to a region of the TRAC locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least The engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 119-138, or comprises a sequence having 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% identity to any one of SEQ ID NOS: 119-138. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease having at least 55% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO: 722. In some embodiments, the engineered guide RNA comprises a sequence having 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% identity to any one of SEQ ID NOs: 121, 132, 136, 130, 134, 135, or 137, or a sequence having at least 80% identity to the targeting sequence of any one of SEQ ID NOs: 121, 132, 136, 130, 134, 135, or 137. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9A. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0029] In some embodiments, the disclosure provides methods of disrupting a B2M locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the B2M locus, and wherein the engineered guide RNA hybridizes to a region of SEQ ID NOs: 185-210 by 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135 The engineered guide RNA is configured to hybridize to or comprises a targeting sequence having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 159-184, or comprises a sequence having 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% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease comprising 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722.In some embodiments, the engineered guide RNA has 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% identity to any one of SEQ ID NOs: 159, 165, 168, 174, or 184. or a sequence having 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% identity to the targeting sequence of any one of SEQ ID NOs: 159, 165, 168, 174, or 184. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9B. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0030] In some aspects, the present disclosure provides a method of disrupting the TRBC1 locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC1 locus, and the engineered guide RNA hybridizes to a region of SEQ ID NOs: 252-292 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%, The engineered guide RNA may be configured to hybridize to or comprise a targeting sequence having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:211-251, or may comprise a sequence having 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% identity to any one of SEQ ID NOs:211-251. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease comprising 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722. In some embodiments, the engineered guide RNA comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 211, 212, 215, 241, or 242, or comprises a targeting sequence having 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% identity to the targeting sequence of any one of SEQ ID NOs: 211, 212, 215, 241, or 242. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9C.In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0031] In some aspects, the disclosure provides a method of disrupting the TRBC2 locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the TRBC2 locus, and the engineered guide RNA hybridizes to a region of the TRBC2 locus that is 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%, or at least 93% of the sequence set forth in SEQ ID NOs: 338-382. The engineered guide RNA may be configured to hybridize to or comprise a targeting sequence having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 293-337, or may comprise a sequence having 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% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease comprising 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722.In some embodiments, the engineered guide RNA has 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% identity to any one of SEQ ID NOs: 296, 306, or 332. or comprises a targeting sequence having 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% identity to the targeting sequence of any one of SEQ ID NOs: 296, 306, or 332. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9C. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0032] In some aspects, the disclosure provides methods of disrupting the ANGPTL3 locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the ANGPTL3 locus, wherein the engineered guide RNA hybridizes to a targeting sequence having at least 80% identity to SEQ ID NOs: 478-572. The guide RNA configured to, comprising, or engineered to guide a nucleotide sequence comprises a sequence having 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% identity to any one of SEQ ID NOs: 383-477. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722.In some embodiments, the engineered guide RNA is 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% identical to any one of SEQ ID NOs: 419, 425, 431, 439, 447, 453, 461, 467, 471, or 473. In some embodiments, the engineered guide RNA comprises a sequence having identity or 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% identity to any one of SEQ ID NOs: 419, 425, 431, 439, 447, 453, 461, 467, 471, or 473. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9D. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0033] In some aspects, the disclosure provides methods of disrupting the PCSK9 locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the PCSK9 locus, and wherein the engineered guide RNA hybridizes to a region of the PCSK9 locus that is 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138 The engineered guide RNA may be configured to hybridize to or comprise a targeting sequence having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 573-587, or may comprise a sequence having 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% identity to any one of SEQ ID NOs: 573-587. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease comprising 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722. In some embodiments, the engineered guide comprises a sequence having 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% identity to any one of SEQ ID NOs: 574, 578, 581, or 585. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 9E. In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid.In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0034] In some embodiments, the disclosure provides methods of disrupting an albumin locus in a cell, the method comprising introducing into the cell (a) any of the endonucleases described herein; and (b) an engineered guide RNA, wherein the engineered guide RNA is configured to form a complex with the endonuclease, wherein the engineered guide RNA comprises a targeting sequence configured to hybridize to a region of the albumin locus, and wherein the engineered guide RNA hybridizes to a region of the albumin locus that is 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 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 181%, at least 182%, at least 183%, at least 184%, at least 185%, at least 186%, at least 187%, at least 188%, at least 189%, at least 190%, at least 200%, at least 210%, at least 211%, at least 212%, at least 213%, at least 214%, at least 215%, at least 216%, at least 217%, at least 218%, at least 219%, at least 220%, at least 221%, at least 222%, at least 223%, at least 224%, at least 225%, at least 226%, at least 227%, at least 228%, at least 229%, at least 230%, at least 231%, at least 232%, at least 233%, at least 234%, at least 235%, at least 236%, at least 237%, at least 238%, at least 239%, at least 240%, or the engineered guide RNA comprises a targeting sequence having 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% identity to the targeting sequence of any one of SEQ ID NOs: 67-86 or 646-695. In some embodiments, the endonuclease is a Class 2, Type II Cas endonuclease. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the endonuclease comprises any of the fusion or engineered endonucleases described herein. In some embodiments, the Class 2, Type II Cas endonuclease comprises any of the Type II Cas endonucleases described herein.In some embodiments, the Class 2, Type II Cas endonuclease comprises a fusion endonuclease 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% identity to SEQ ID NO: 10 or a variant thereof. In some embodiments, the engineered guide RNA comprises a sequence having 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 the non-degenerate nucleotides of SEQ ID NO:722. In some embodiments, the engineered guide RNA is complementary to or comprises a sequence having 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% identity to any one of SEQ ID NOs: 67, 68, 70, 71, 72, 76, 79, 80, 647, 648, 649, 653, 654, 655, 656, 673, 680, 681, or 682. In some embodiments, the engineered guide RNA comprises the nucleotide sequence of any one of the guide RNAs from Table 6.In some embodiments, introducing into the cell further comprises contacting the cell with a nucleic acid or vector encoding the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the vector or nucleic acid. In some embodiments, introducing into the cell further comprises contacting the cell with a ribonucleoprotein complex (RNP) comprising the fusion protein or guide polynucleotide, or comprises contacting the cell with a lipid nanoparticle (LNP) comprising the RNP.

[0035] In some aspects, the present disclosure provides an endonuclease comprising an engineered amino acid sequence having at least 55% sequence identity to any one of SEQ ID NOs: 1-27, 108, or 109-110.

[0036] In some aspects, the present disclosure provides an engineered nuclease system comprising an endonuclease described herein and an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the guide ribonucleic acid structure comprising a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the endonuclease is derived from an uncultured microorganism. In some embodiments, the endonuclease is not Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease. In some embodiments, the endonuclease has less than 86% identity to the SpyCas9 endonuclease. 2+ The present invention further comprises a source of

[0037] In some aspects, the disclosure provides an engineered nuclease comprising: (a) a Class 2, Type II Cas enzyme RuvC and HNH domain having at least 55% sequence identity to the RuvC and HNH domain of any one of SEQ ID NOs: 1-27, 108, or 109-110; and (b) a Class 2, Type II Cas enzyme PAM-interacting (PI) domain having at least 55% sequence identity to the PAM-interacting (PI) domain of any one of SEQ ID NOs: 1-27, 108, or 109-110. In some embodiments, (a) and (b) do not occur together in nature. In some embodiments, the Class 2, Type II Cas enzyme is derived from an uncultured microorganism. In some embodiments, the endonuclease has less than 86% identity to SpyCas9 endonuclease. In some embodiments, the engineered nuclease comprises a sequence having at least 55% sequence identity to any one of SEQ ID NOs: 1-27.

[0038] In some aspects, the present disclosure provides an engineered nuclease system comprising an endonuclease according to any of the aspects or embodiments described herein and an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, the guide ribonucleic acid structure comprising a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence and a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the guide ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOS: 28-32 or 33-44, or a variant thereof. In some embodiments, the system further comprises a PAM sequence compatible with the nuclease adjacent to the target nucleic acid site. In some embodiments, the PAM sequence is located 3' of the target deoxyribonucleic acid sequence.

[0039] In some embodiments, the present disclosure provides an engineered single molecule heterologous guide polynucleotide compatible with a Class 2, Type II enzyme according to any aspect or embodiment described herein, wherein the heterologous guide polynucleotide comprises a chemical modification set forth in any one of SEQ ID NOs: 605-610 or 1789-1865.

[0040] In some aspects, the present disclosure provides a method of targeting an albumin gene, comprising introducing into a cell a system according to any one of the aspects or embodiments described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to a sequence comprising any one of SEQ ID NOs: 67-86.

[0041] In some aspects, the disclosure provides a method of targeting the HAO1 gene, comprising introducing into a cell a system according to any one of the aspects or embodiments described herein, wherein the guide ribonucleic acid sequence is configured to hybridize to any one of SEQ ID NOs: 611-633. In some embodiments, the guide ribonucleic acid sequence is configured to hybridize to any one of SEQ ID NOs: 615, 618, 620, 624, or 626. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOs: 605-610 or 1789-1865. In some embodiments, the guide ribonucleic acid comprises a sequence according to any one of SEQ ID NOs: 1812-1824, 1835, 1849, 1858, or 1861.

[0042] In some aspects, the present disclosure provides a cell comprising an endonuclease described herein. In some aspects, the present disclosure provides a cell comprising any of the nucleic acid molecules described herein. In some aspects, the present disclosure provides a cell comprising any of the engineered nuclease systems described herein.

[0043] In certain embodiments, described herein are engineered endonucleases comprising: a) an N-terminal portion comprising a sequence having at least 80% sequence identity to SEQ ID NO:696; and b) a C-terminal portion comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs:697-721. In some embodiments, the N-terminal portion and the C-terminal portion are directly fused to each other. In some embodiments, the N-terminal portion and the C-terminal portion are joined by a linker. In some embodiments, the linker is a glycine- and / or serine-rich linker, a large protein domain, a long helix, or a short helix. In some embodiments, the linker is (GGGGS)n, where n is an integer between 1 and 20. In some embodiments, the linker is GGGGS (SEQ ID NO:2864). In some embodiments, the N-terminal portion comprises a sequence having at least 90% sequence identity to SEQ ID NO:696. In some embodiments, the N-terminal portion comprises a sequence having 100% sequence identity to SEQ ID NO:696. In some embodiments, the C-terminal portion comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 697-721. In some embodiments, the C-terminal portion comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of SEQ ID NOs: 60-66, 117, 865-919, and 2855-2863.

[0044] In certain embodiments, described herein are engineered endonucleases comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862.

[0045] In certain embodiments, described herein is an engineered endonuclease comprising a sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the engineered endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to SEQ ID NO: 10.

[0046] In certain embodiments, described herein are engineered endonucleases comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 109-110 and 2842-2854.

[0047] 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: 1-27 and 771-862; 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: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862. 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: 28-45, 605-610, 646-695, 863, and 1789-1826. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 28-45, 605-610, 646-695, 863, and 1789-1826. In some embodiments, the fused endonuclease is configured to bind to a PAM comprising any one of SEQ ID NOs: 60-66, 865-919, and 2863.

[0048] 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: 109-110 and 2842-2854; 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: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the engineered endonuclease is non-covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is fused to the engineered guide polynucleotide. In some embodiments, the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 111-113. In some embodiments, the engineered guide polynucleotide comprises a sequence having 100% sequence identity to any one of SEQ ID NOs: 111-113. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of SEQ ID NOs: 117 and 2855-2862.

[0049] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 albumin gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 67-86.

[0050] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 TRAC gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.

[0051] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 B2M gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 159-184. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 185-210.

[0052] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 211-251. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 252-292.

[0053] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 TRBC2 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 293-337. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 338-382.

[0054] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.

[0055] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 PCSK9 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391.

[0056] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770.

[0057] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 AAVS1 locus or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087.

[0058] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 GPR146 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1657-1725.

[0059] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 APOA1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.

[0060] In certain embodiments, described herein is an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 HAO1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.

[0061] In certain embodiments, described herein are methods for modifying a target nucleic acid sequence, the method comprising contacting the target nucleic acid sequence with an endonuclease described herein or an engineered nuclease system described herein. In some embodiments, modifying the target nucleic acid sequence comprises binding, nicking, or cleaving the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence 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. In some embodiments, the gRNA is encoded by a sequence having any one of SEQ ID NOs: 251-260, 271-274, and 279-290. In some embodiments, the target nucleic acid sequence comprises a sequence having any one of SEQ ID NOs: 261-270, 275-278, and 291-302.

[0062] In certain embodiments, described herein are methods for modifying a target nucleic acid sequence in a mammalian cell, the methods comprising contacting the mammalian cell with an endonuclease described herein or an engineered nuclease system described herein. In some embodiments, the method further comprises selecting the cell containing the modification.

[0063] In certain embodiments, described herein is a method of modifying an albumin gene, the method comprising contacting the albumin 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: 1-27 and 771-862; 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 albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 67-86.

[0064] In certain embodiments, described herein are methods of modifying a TRAC gene, the method comprising contacting the TRAC 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: 1-27 and 771-862; 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 TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.

[0065] In certain embodiments, described herein are methods of modifying a B2M gene, the method comprising contacting the B2M 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: 1-27 and 771-862; 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 B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 159-184. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 185-210.

[0066] In certain embodiments, described herein are methods for modifying the TRBC1 gene, comprising contacting the TRBC1 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: 1-27 and 771-862; 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 TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOS: 211-251. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOS: 252-292.

[0067] In certain embodiments, described herein are methods for modifying the TRBC2 gene, comprising contacting the TRBC2 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: 1-27 and 771-862; 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 TRBC2 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOS: 293-337. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOS: 338-382.

[0068] In certain embodiments, described herein is a method of modifying the 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: 1-27 and 771-862; 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 endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.

[0069] In certain embodiments, described herein are methods of modifying a PCSK9 gene, comprising contacting the PCSK9 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: 1-27 and 771-862; 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 PCSK9 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391.

[0070] In certain embodiments, described herein are methods of modifying a VCP gene, the method comprising contacting the VCP gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770.

[0071] In certain embodiments, described herein are methods of modifying the AAVS1 locus, the method comprising contacting the AAVS1 locus 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: 1-27 and 771-862; 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 AAVS1 locus or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087.

[0072] In certain embodiments, described herein are methods of modifying the GPR146 gene, comprising contacting the GPR146 gene with an engineered nuclease system comprising: a) an engineered endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-27 and 771-862; 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 GPR146 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1657-1725.

[0073] In certain embodiments, described herein are methods of modifying an APOA1 gene, the method comprising contacting the APOA1 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: 1-27 and 771-862; 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 APOA1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.

[0074] In certain embodiments, described herein is a method of modifying a TRAC gene, the method comprising contacting the TRAC 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: 1-27 and 771-862; 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 HAO1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.

[0075] In certain embodiments, described herein are cells comprising an endonuclease described herein or an engineered nuclease system 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 cells are engineered cells. In some embodiments, the cells are stable cells.

[0076] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. [Brief explanation of the drawings]

[0077] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Figure 1A]

[0023] Figure 1A shows the natural PAM specificity of the various effectors described herein. Figure 1A shows a phylogenetic tree of the various effectors described herein. [Figure 1B] Figure 1B shows the native PAM specificities of various effectors described herein. Figure 1B is a table of the PAM specificities of native RNA-guided CRISPR-associated endonucleases. [Figure 2] Illustrates the concept of domain swapping between RNA-guided CRISPR-associated nucleases. [Figure 3A] Multiple sequence alignments are shown to guide optimal breakpoint determination. Figure 3A shows SaCas9 and SpCas9 aligned to several proteins described herein, identifying the conserved residue (alanine residue) at the end of these sequences as the proposed C-terminus of the exchanged section. [Figure 3B] Figure 3B shows the C-terminal domain of the SaCas9 protein, spanning the swapped RuvC-III, WED, TOPO, and CTD domains. The PAM-interacting domain is composed of the TOPO and CTD domains. The active site residues (D10, E477, and H701 in the RuvC domain and D556, D557, and N580 in the NHN domain) are not included in the swapped C-terminal domain. [Figure 4] Screening of chimeras using an in vitro PAM enrichment assay when MG3-6 was recombined with various C-terminal domains from closely and distantly related nucleases. sgRNAs derived from the N-terminal parent domains were used for RNA-guided nuclease activity. [Figure 5A]The PAM sequence of the functional chimera described herein (Figure 5A) and a sequence logo representation of the PAM sequence (Figure 5B) are shown. Given the breakpoint exchange of the predicted C-terminal domains of RuvC-III, WED, TOPO, and CTD, the chimera was functional when recombined with a closely related nuclease. The engineered chimeras tended to preserve the PAM specificity derived from the PAM-interacting domain of the native protein, even when the native protein was not functional in the same experiment. [Figure 5B] The PAM sequence of the functional chimera described herein (Figure 5A) and a sequence logo representation of the PAM sequence (Figure 5B) are shown. Given the breakpoint exchange of the predicted C-terminal domains of RuvC-III, WED, TOPO, and CTD, the chimera was functional when recombined with a closely related nuclease. The engineered chimeras tended to preserve the PAM specificity derived from the PAM-interacting domain of the native protein, even when the native protein was not functional in the same experiment. [Figure 6] Screening of chimeras using an in vitro PAM enrichment assay using chimeras incorporating MG3-6 with various C-terminal domains from closely and distantly related nucleases is shown. sgRNAs derived from the C-terminal parent domains were used for RNA-guided nuclease activity. Numbers in parentheses indicate the sgRNA species. Use of sgRNAs derived from the C-terminal parent domains did not rescue activity. [Figure 7] The predicted structures of MG3-6 and MG15-1 are shown. The WED and PI domains of MG3-6 were exchanged with those of the MG15-1 counterparts to generate Chimera 1 (C1). Alternatively, the PI domain of MG3-6 was exchanged with that of the MG15-1 counterpart to generate Chimera 2 (C2). [Figure 8A]The results of in vitro PAM enrichment assays and Sanger sequencing for PAM specificity are shown. C1: MG3-6 + MG15-1(WP) and C2: MG3-6 + MG15-1(P). The engineered chimeras tend to preserve the PAM specificity derived from the PAM-interacting domain of the native protein. PAM enrichment assays were performed in triplicate. (Figure 8A) shows an agarose gel representation of the assay demonstrating that the sequence was cleaved in the presence of active enzyme, and (Figure 8B) shows a sequence logo representation of the PAM sequence determined by the assay. [Figure 8B] The results of in vitro PAM enrichment assays and Sanger sequencing for PAM specificity are shown. C1: MG3-6 + MG15-1(WP) and C2: MG3-6 + MG15-1(P). The engineered chimeras tend to preserve the PAM specificity derived from the PAM-interacting domain of the native protein. PAM enrichment assays were performed in triplicate. (Figure 8A) shows an agarose gel representation of the assay demonstrating that the sequence was cleaved in the presence of active enzyme, and (Figure 8B) shows a sequence logo representation of the PAM sequence determined by the assay. [Figure 9A] Figure 9 shows the activity of the chimeras described herein in mammalian cells. mRNA hybridization to the chimeras was co-transfected with 20 different sgRNAs (see, e.g., SEQ ID NOS: 67-86) into Hepa 1-6 cells. Editing was assessed by Sanger sequencing and inference of CRISPR editing (ICE). Figure 9A shows the editing efficiency of the guides tested. Two biological replicates are shown. [Figure 9B] Figure 9B shows the activity of the chimeras described herein in mammalian cells. mRNA hybridization to the chimera was co-transfected with 20 different sgRNAs (see, e.g., SEQ ID NOS: 67-86) into Hepa 1-6 cells. Editing was assessed by Sanger sequencing and inference of CRISPR editing (ICE). Figure 9B shows the indel profile generated by a representative guide. [Figure 10] Results of guide screening in Hepa1-6 cells are shown, where guides were delivered as mRNA and gRNA using Lipofectamine Messenger Max. [Figure 11A] The structural parts of the MG3-6 / 3-4 guide are shown. [Figure 11B] The structural parts of the MG3-6 guide are shown. [Figure 12] Figure 1 shows the activity of chemically modified MG3-6 / 3-4 guides in Hepa1-6 cells when delivered as mRNA and gRNA using Lipofectamine Messenger Max. [Figure 13] 1 shows the stability of chemically modified MG3-6 / 3-4 guides over 9 hours at 37° C. [Figure 14] 1 shows the stability of chemically modified MG3-6 / 3-4 guides over 21 hours at 37° C. [Figure 15A] Figure 15A shows an in vitro screening of type VA chimeras. Figure 15A shows an agarose gel of the amplified cleavage products for each cleavage reaction. Positive enrichment is observed for the MG29-1 + MG29-5 chimeras, which are domain-swapped (numbers in parentheses indicate sgRNA species) from the same family. [Figure 15B] Figure 15B shows the in vitro screening of type VA chimeras. Figure 15B shows the sequence logo representation of the PAM for the parent enzyme and chimeras derived therefrom. [Figure 16] This shows the results of gene editing at the DNA level of TRAC in HEK293T cells. [Figure 17] This shows the results of gene editing at the DNA level of B2M in HEK293T cells. [Figure 18] Figure 1 shows the results of gene editing at the DNA and phenotype levels of TRAC in T cells. [Figure 19] This shows the results of gene editing at the DNA level of B2M in T cells. [Figure 20] 1 shows the results of gene editing at the phenotypic level of TRBC1 and TRBC2 in T cells. [Figure 21] This shows the results of gene editing at the DNA level of ANGPTL3 in Hep3B cells. [Figure 22] This shows the results of gene editing at the DNA level of PCSK9 in Hep3B cells. [Figure 23] 1 shows genome editing at the HAO-1 locus by MG3-6 / 3-4 in wild-type mice analyzed by next-generation sequencing. [Figure 24] 1 shows glycolate oxidase protein levels in the liver of mice treated with MG3-6 / 3-4 mRNA and guide RNA targeting the HAO-1 gene. [Figure 25] 1 shows genome editing at the HAO-1 locus in wild-type mice treated with MG3-6 / 3-4 mRNA and guide RNA 7 (G7) targeting HAO-1 with four different chemical modifications. [Figure 26] 1 shows Western blot analysis of glycolate oxidase (GO) / HAO-1 protein levels in the liver of mice 11 days after treatment with LNPs encapsulating MG3-6 / 3-4 mRNA and sgRNA 7 (G7) with four different chemical modifications, as described in Example 19. Individual mice are indicated by numbers. [Figure 27] 10 shows the activity of chemically modified guides in Hep3B cells when delivered as mRNA and gRNA using lipid transfection to target HAO-1 as in Example 20. [Figure 28] 1 shows the activity of chemically modified guides in primary human hepatocytes when delivered as mRNA and gRNA using lipid transfection to target HAO-1 as described in Example 21. [Figure 29] 1 shows the demonstration of mammalian cell editing and determination of PAM sequences for two MG3-6 chimeric enzymes, as evaluated in Example 22. [Figure 30] 1 shows evaluation of MG3-6 / 3-4 guide chemistries for editing activity in mouse liver after in vivo delivery in LNPs as in Example 23. [Figure 31] 1 shows the results of gene editing at the DNA level for VCP in K562 cells as in Example 24. [Figure 32]

[0039] Figure 1 shows gene editing results at the DNA level in K562 cells. Guides were designed to target TRAC and AAVS1. MG3-6_3-3RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-3. [Figure 33]

[0033] Figure 1 shows gene editing results at the DNA level in K562 cells. Guides were designed to target TRAC and AAVS1. MG3-6_3-7RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-7. [Figure 34]

[0023] Figure 1 shows gene editing results at the DNA level in K562 cells. The guide was designed to target TRAC. MG3-6_3-8RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-8. [Figure 35]

[0023] Figure 1 shows gene editing results at the DNA level in K562 cells. The guide was designed to target TRAC. MG3-6_3-8RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-8. [Figure 36]

[0023] Figure 1 shows the results of gene editing at the DNA level in human Hep3B cells. The guide was designed to target PCSK9. MG3-6_3-4RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-4. [Figure 37]

[0023] Figure 1 shows the results of gene editing at the DNA level in human Hep3B cells. The guide was designed to target ANGPTL3. MG3-6_3-4RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-4. [Figure 38] This shows the results of gene editing in human Hep3B cells at the DNA level. The guide was designed to target APOA1. MG3-6_3-8RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-8. [Figure 39]

[0023] Figure 1 shows gene editing results at the DNA level in mouse Hepa1-6 cells. The guide was designed to target GPR146. MG3-6_3-7RWP is a chimera that replaces the partial RuvC-III, WED, and PID of MG3-6 with MG3-7RWP. [Figure 40]

[0023] Figure 1 shows gene editing results at the DNA level in mouse Hepa1-6 cells. The guide was designed to target APOA1. MG3-6_3-4RWP is a chimera that exchanges the partial RuvC-III, WED, and PID of MG3-6 with MG3-4. [Figure 41] The design strategy of the MG3-6 chimera is shown. Panel (a) (top left) of Figure 41 shows the predicted domain boundaries of MG3-6 and the breakpoints of recombination with MG3 and MG150 members. Panel (b) (bottom left) of Figure 41 shows the predicted 3-D structure of MG3-6 and the breakpoints of protein recombination. The structure is predicted by Novafold. Panel (c) (right) of Figure 41 shows a multiple sequence alignment (MSA) of MG3 and MG150 members. The breakpoints are indicated by arrows. [Figure 42A] Screening of MG3-6 chimeras and characterization of PAM specificity. Figure 42A (left) shows DNA agarose gel electrophoresis analysis assessing enzyme activity. [Figure 42B] Screening of MG3-6 chimeras and characterization of PAM specificity. Figure 42B (right) shows the phylogenetic tree and sequence logos for functional chimeras. [Figure 43-1] The design strategy of the MG3-6_3-8 chimera is shown. Panel (a) (top left) of Figure 43 shows the predicted domain boundaries of MG3-6_3-8 and the breakpoints of recombination with MG3 and MG150 members. Panel (b) (bottom left) of Figure 43 shows the predicted 3-D structure of MG3-6 and the breakpoints of protein recombination. The structure is predicted by Novafold. [Figure 43-2]The design strategy for the MG3-6_3-8 chimera is shown. Panel (c) (center) of Figure 43 shows a multiple sequence alignment (MSA) of the MG3 and MG150 members. Panel (d) (right) of Figure 43 shows a multiple sequence alignment (MSA) of the MG15 members. Breakpoints are indicated by arrows. [Figure 44A] Screening of MG3-6_3-8 chimeras and characterization of PAM specificity. Figure 44A (top) shows DNA agarose gel electrophoresis analysis assessing enzyme activity. [Figure 44B-1] Screening of MG3-6_3-8 chimeras and characterization of PAM specificity. Figure 44B (bottom) shows the sequence logo consensus representation of the PAM sequences for functional chimeras. [Figure 44B-2] Screening of MG3-6_3-8 chimeras and characterization of PAM specificity. Figure 44B (bottom) shows the sequence logo consensus representation of the PAM sequences for functional chimeras. [Figure 45] 1 shows the structures of exemplary cationic lipids that can be used in the lipid nanoparticles described herein. [Figure 46] 1 shows evaluation of MG3-6 / 3-4 guide chemistries for HAO-1 gene editing and mRNA knockdown activity in mouse liver after in vivo delivery in LNP as described in Example 28. [Figure 47] 1 shows the results of gene editing at the DNA level for mApoa1 in Hepa1-6 cells. [Figure 48] 1 shows the results of gene editing at the DNA level for mAngptl3 in Hepa1-6 cells. [Figure 49] 1 shows the results of gene editing at the DNA level for mTrac in Hepa1-6 cells. [Figure 50] 1 shows genome editing at the APOA1 and ANGPTL3 loci by MG3-6 / 3-8 in wild-type mice analyzed by next-generation sequencing. [Figure 51A] 1 shows a phylogenetic tree of the various MG29 effectors described herein. [Figure 51B] The three-dimensional structure of the MG29-1 effector predicted using Alphafold2 is shown. The nucleic acid forming the R-loop is modeled via structural alignment with Cpf1 (PDB ID: 5XH7). The REC, RuvC, and NUC domains are colored gray, cyan, and pink, respectively. The PAM-interacting domains, i.e., WED-II, WED-III, and PID domains, are colored yellow and orange, respectively. [Figure 52A] Phylogenetic analysis of type VA nucleases. Phylogenetic trees were inferred with RAxML from the MAFFT global (g-ins-i) multiple sequence alignment. Ancestral WedII, PI, and WedIII domains were generated for three ancestral nodes (nodes highlighted with black circles: MG29-229, MG29-230, and MG29-231). [Figure 52B] The 3D structure prediction of the ancestral domain from MG29-28 is shown relative to the predicted structure of MG29-1. The high similarity of these domains is observed by the overlap between the structures (top) and the conservation of key residues in the sequence alignment (bottom). Key residues involved in PAM binding are indicated by the dark box above the alignment and by stick cartoon visualization in the 3D alignment. [Figure 53A] Figure 53A shows a multiple sequence alignment of MG29-1 homologs to determine optimal breakpoints for chimera formation. Figure 53A shows MG29-1 and FnCas12a aligned to several proteins described herein. The WED-II, PID, and WED-III domains are annotated to highlight the domains that have been exchanged. Dashed lines indicate the cleavage sites for exchanging these enzymatic domains. Overall, the exchanged regions span the WED-II, PID, and WED-III domains. [Figure 53B]Figure 53B shows multiple sequence alignment of MG29-1 homologs to determine optimal breakpoints for chimera formation. Figure 53B: The C-termini of the MG29-1 protein and its homologs consist of breakpoints at the edges of the REC domain and the WED-II domain. Overall, the exchanged regions span the WED-II, PID, and WED-III domains. [Figure 53C] Figure 53C shows multiple sequence alignment of MG29-1 homologs to determine optimal breakpoints for chimera formation. Figure 53C: The N-termini of the MG29-1 protein and its homologs consist of breakpoints at the edges of the WED-III and RuvC domains. Overall, the exchanged regions span the WED-II, PID, and WED-III domains. [Figure 54] Figure 1 shows in vitro screening of type VA chimeras. The agarose gel shows the amplified cleavage products for each cleavage reaction. Positive enrichment was observed for chimeras with MG29-1 and several of its homologs, i.e., MG29-13, MG29-14, MG29-16, MG29-20, and MG29-18. [Figure 55] Figure 1 shows in vitro screening of the type VA ancestral sequence and its corresponding chimeras with MG29-1. The agarose gel shows the amplified cleavage products for each cleavage reaction. Positive enrichment was observed only for the ancestrally reconstructed MG29-230 and MG29-231. However, all corresponding chimeras were found to be active. [Figure 56-1] PAMs associated with MG29-1 and chimeras derived from it are shown. [Figure 56-2] PAMs associated with MG29-1 and chimeras derived from it are shown.

[0078] Brief description of the sequence listing The Sequence Listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions, and systems according to the present disclosure. Below are illustrative examples of sequences therein.

[0079] MG3-6 Chimera SEQ ID NOs: 1 to 27 and 771 to 862 show the full-length peptide sequences of the MG3-6 chimeric nuclease.

[0080] SEQ ID NO: 108 shows the nucleotide sequence of the MG3-6 / 3-4 nuclease, including the 5'UTR, NLS, CDS, NLS, 3'UTR, and polyA tail.

[0081] SEQ ID NO: 722 shows the nucleotide sequence of the MG3-6 / 3-4 guide sgRNA scaffold.

[0082] SEQ ID NOs: 28-45, 605-610, 646-695, 863, and 1789, 1826 show the nucleotide sequences of sgRNAs engineered to function with the MG3-6 chimeric nuclease.

[0083] SEQ ID NO: 603 shows the DNA coding sequence of MG3-6 / 3-4.

[0084] SEQ ID NO: 604 shows the protein sequence of the MG3-6 / 3-4 cassette coding sequence.

[0085] MG29-1 Chimera SEQ ID NOs: 109 to 110 and 2842 to 2854 show the full-length peptide sequences of the MG29-1 chimeric nuclease.

[0086] SEQ ID NOs: 111-113 show the nucleotide sequences of sgRNAs engineered to function with the MG29-1 chimeric nuclease.

[0087] MG Chimera SEQ ID NO: 696 shows the N-terminal peptide sequence (1-742) of MG3-6.

[0088] SEQ ID NOs: 697 to 721 show C-terminal peptide sequences.

[0089] Albumin targeting SEQ ID NOs: 67-86 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target albumin.

[0090] TRAC targeting SEQ ID NOs: 119-138 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target TRAC.

[0091] SEQ ID NOs: 139 to 158 show the DNA sequences of the TRAC target sites.

[0092] SEQ ID NOs: 922-924 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-3 nuclease to target TRAC.

[0093] SEQ ID NOs: 925 to 927 show the DNA sequences of the TRAC target sites.

[0094] SEQ ID NOs: 972-991 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-7 nuclease to target TRAC.

[0095] SEQ ID NOs: 992 to 1011 show the DNA sequences of the TRAC target sites.

[0096] SEQ ID NOs: 1088-1183, 1280-1320, 2390-2485, and 2582-2617 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-8 nuclease to target TRAC.

[0097] SEQ ID NOs: 1184 to 1279, 1321 to 1361, 2486 to 2581, and 2618 to 2653 show the DNA sequences of the TRAC target sites.

[0098] B2M targeting SEQ ID NOs: 159-184 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target B2M.

[0099] SEQ ID NOs: 185 to 210 show the DNA sequences of the B2M target sites.

[0100] TRBC1 targeting SEQ ID NOs: 211-251 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target TRBC1.

[0101] SEQ ID NOs: 252 to 292 show the DNA sequences of the TRBC1 target sites.

[0102] TRBC2 targeting SEQ ID NOs: 293-337 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target TRBC2.

[0103] SEQ ID NOs: 338 to 382 show the DNA sequences of the TRBC2 target sites.

[0104] ANGPTL3 targeting SEQ ID NOs: 383-477 and 1392-1489 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target ANGPTL3.

[0105] SEQ ID NOs: 478 to 572 and 1490 to 1587 show the DNA sequences of the ANGPTL3 target sites.

[0106] SEQ ID NOs: 2120-2215 and 2312-2350 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-8 nuclease to target ANGPTL3.

[0107] SEQ ID NOs: 2216 to 2311 and 2351 to 2389 show the DNA sequences of the ANGPTL3 target sites.

[0108] PCSK9 targeting SEQ ID NOs: 573-587 and 1362-1376 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target PCSK9.

[0109] SEQ ID NOs: 588 to 602 and 1377 to 1391 show the DNA sequences of PCSK9 target sites.

[0110] VCP R155 targeting SEQ ID NOs: 723-738 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-8 nuclease to target VCP R155.

[0111] SEQ ID NOs: 739 to 754 show the DNA sequences of the VCPR155 target site.

[0112] SEQ ID NOs: 755-762 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target VCP R155.

[0113] SEQ ID NOs: 763 to 770 show the DNA sequences of the VCPR155 target site.

[0114] AAVS1 targeting SEQ ID NOs: 928-949 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-3 nuclease to target AAVS1.

[0115] SEQ ID NOs: 950 to 971 show the DNA sequences of the AAVS1 target site.

[0116] SEQ ID NOs: 1012-1049 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-7 nuclease to target AAVS1.

[0117] SEQ ID NOs: 1050 to 1087 show the DNA sequences of the AAVS1 target sites.

[0118] GPR146 targeting SEQ ID NOs: 1588-1656 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-7 nuclease to target M. musculus GPR146.

[0119] SEQ ID NOs: 1657 to 1725 show the DNA sequences of the M. musculus GPR146 target site.

[0120] APOA1 targeting SEQ ID NOs: 1726-1744 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target M. musculus APOA1.

[0121] SEQ ID NOs: 1745 to 1763 show the DNA sequences of the M. musculus APOA1 target site.

[0122] SEQ ID NOs: 1764-1774 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target H. sapiens APOA1.

[0123] SEQ ID NOs: 1775 to 1785 show the DNA sequences of the H. sapiens APOA1 target site.

[0124] SEQ ID NOs: 1866-1961 and 2058-2088 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-8 nuclease to target mouse APOA1.

[0125] SEQ ID NOs: 1962 to 2057 and 2089 to 2119 show the DNA sequences of mouse APOA1 target sites.

[0126] HAO1 targeting SEQ ID NOs: 611-633 and 1789-1826 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target M. musculus HAO1.

[0127] SEQ ID NOs: 1827-1865 show the nucleotide sequences of sgRNAs engineered to function with MG3-6 / 3-4 nuclease to target human HAO1.

[0128] Other arrays SEQ ID NOs: 87 to 102, 118, 634 to 645, and 1786 to 1787 represent primer sequences.

[0129] SEQ ID NOs: 103 to 107, 920 to 921, and 1788 show the plasmid sequences. DETAILED DESCRIPTION OF THE INVENTION

[0130] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0131] The practice of some methods disclosed herein employs, unless otherwise indicated, techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds.); the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J.MacPherson, B.D.Hames, and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988), Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0132] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "comprising," "including," "having," "having," or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0133] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0134] As used herein, "cell" refers to a biological cell. A cell can be the basic structural, functional, or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-celled eukaryotic cells, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, liverworts, and mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, etc.), and the like. C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from vertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. In some cases, the cells are not derived from a naturally occurring organism (e.g., the cells may be synthetically produced, sometimes referred to as artificial cells).

[0135] As used herein, the term "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled, such as by using a moiety containing an optically detectable moiety (e.g., a fluorophore). Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL.; and Boehringer 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 Mannheim, Indianapolis, Ind.; and Molecular Examples of chromosomal labeled nucleotides available from Probes, Eugene, Oreg. include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Nucleotides may also be labeled or marked by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can 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).

[0136] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-, double-, or multiple-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. When referring to a polynucleotide, T means U (uracil) in RNA and T (thymine) in DNA. Polynucleotides may have any three-dimensional structure and may perform any function. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined by binding analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0137] The term "transfection" or "transfected" refers to the introduction of nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule may be a gene sequence encoding an entire protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0138] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by a peptide bond. The term does not denote a particular length of the polymer, and is not intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins and proteins with or without secondary or tertiary structure (e.g., domains). The term also encompasses amino acid polymers modified by any other manipulation, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, oxidation, and conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and unnatural amino acids, which are chemically modified to include non-naturally occurring groups or chemical moieties on the amino acid. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0139] As used herein, "non-naturally occurring" may refer to a nucleic acid or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein. Non-naturally occurring may refer to an affinity tag. Non-naturally occurring may refer to a fusion. Non-naturally occurring may refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, or deletions. A non-naturally occurring sequence may exhibit or encode an activity (e.g., an enzymatic activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, a ubiquitination activity, etc.) that may also be exhibited by the nucleic acid or polypeptide sequence to which the non-naturally occurring sequence is fused. A non-naturally occurring nucleic acid or polypeptide sequence may be linked to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid or polypeptide sequence that encodes the chimeric nucleic acid or polypeptide.

[0140] As used herein, the term "promoter" refers to a regulatory DNA region that controls the transcription or expression of a polynucleotide (e.g., a gene) and may be located adjacent to or overlapping the nucleotide or region of nucleotide at which RNA transcription is initiated. A promoter may contain specific DNA sequences that bind protein factors, often called transcription factors, which promote the binding of RNA polymerase to DNA, thereby resulting in gene transcription. A "basal promoter," also referred to as a "core promoter," may refer to a promoter that contains all the basic and necessary elements to promote the transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters typically, but not necessarily, contain a TATA box and / or a CAAT box.

[0141] As used herein, the term "expression" refers to the process by which a nucleic acid sequence or polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) or by which a transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0142] As used herein, "operably linked," "operable linkage," "operatively linked," or their grammatical equivalents refer to the arrangement of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., such that the action (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element can be, but need not be, of the same type as the action of the first genetic element. For example, two genetic elements are operably linked if movement of the first element causes activation of the second element. For example, a regulatory element, which may include a promoter sequence and / or an enhancer sequence, is operably linked to a coding region if the regulatory element helps to initiate transcription of the coding sequence. There can be intervening residues between the regulatory element and the coding region, so long as this functional relationship is maintained.

[0143] As used herein, "vector" refers to a macromolecule or association of macromolecules that contains or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. A vector generally contains genetic elements, such as regulatory elements, operably linked to a gene to facilitate expression of the gene in a target.

[0144] As used herein, "expression cassette" and "nucleic acid cassette" are used interchangeably to refer to a combination of nucleic acid sequences or elements that are expressed together or that are operably linked for expression. In some cases, an expression cassette refers to a combination of a gene or genes with regulatory elements that are operably linked for expression.

[0145] A "functional fragment" of a DNA or protein sequence refers to a fragment that retains a biological activity (either functional or structural) substantially similar to that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be the ability to affect expression in a manner attributable to the full-length sequence.

[0146] The terms "engineered," "synthetic," and "artificial" are used interchangeably herein to refer to an entity modified by human intervention. For example, the terms can refer to a non-naturally occurring polynucleotide or polypeptide. An engineered peptide can, but need not, have low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity, less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity) to a naturally occurring human protein. For example, the VPR domain and the VP64 domain are synthetic transactivation domains. For example, the VPR domain and the VP64 domain are synthetic transactivation domains. By way of non-limiting examples, nucleic acids may be modified by changing their sequence to one that does not occur in nature, nucleic acids may be modified by ligating them to nucleic acids with which they are not naturally associated so that the ligated product has a function not present in the original nucleic acid, engineered nucleic acids may be synthesized in vitro with sequences that do not occur in nature, proteins may be modified by changing their amino acid sequence to a sequence that does not occur in nature, and engineered proteins may acquire new functions or properties. An "engineered" system comprises at least one engineered component.

[0147] The term "tracrRNA" or "tracr sequence" refers to a transactivating CRISPR RNA. tracrRNA interacts with CRISPR (cr)RNA to form the guide (g)RNA of type II and subtype VB CRISPR-Cas systems. When tracrRNA is engineered, it can have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from S. pyogenes, S. aureus). tracrRNA can also refer to modified forms of tracrRNA, which can contain nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeras. A tracrRNA may refer to a nucleic acid that may be at least about 60% identical to a wild-type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six contiguous nucleotides. For example, a tracrRNA sequence may be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild-type exemplary tracrRNA (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least six contiguous nucleotides. Type II tracrRNA sequences can be predicted on a genomic sequence by identifying regions that have complementarity to portions of repeat sequences in adjacent CRISPR arrays.

[0148] As used herein, "guide nucleic acid" or "guide polynucleotide" refers to a nucleic acid that can hybridize to a target nucleic acid, thereby directing an associated nuclease to the target nucleic acid. A guide nucleic acid can be RNA (guide RNA or gRNA). A guide nucleic acid can be DNA. A guide nucleic acid can be a mixture of RNA and DNA. A guide nucleic acid can include crRNA or tracrRNA, or a combination of both. A guide nucleic acid can be engineered. A guide nucleic acid can be programmed to specifically bind to a target nucleic acid. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid can be referred to as the complementary strand. A strand of a double-stranded target polynucleotide that is complementary to the complementary strand and therefore not complementary to the guide nucleic acid can be referred to as the non-complementary strand. A guide nucleic acid can comprise a polynucleotide strand and can be referred to as a "single guide nucleic acid." A guide nucleic acid can comprise two polynucleotide strands and can be referred to as a "dual guide nucleic acid." Otherwise, the term "guide nucleic acid" may be generic, referring to both single and double guide nucleic acids. A guide nucleic acid may include a segment that may be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence" or "spacer." A nucleic acid targeting segment may include a subsegment that may be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment."

[0149] As used herein, the terms "gene editing" and "genome editing" may be used interchangeably. Gene editing or genome editing refers to changing the nucleic acid sequence of a gene or genome. Genome editing can include, for example, insertions, deletions, and mutations.

[0150] The term "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence over a local or global comparison window, as measured using a sequence comparison algorithm. Suitable sequence comparison algorithms for polypeptide sequences include, for example, BLASTP using the BLOSUM62 scoring matrix setting parameters of word length (W) of 3, expectation (E) of 10, and presence of 11, gap cost at extension of 1, and using a conditional composition score matrix adjustment for polypeptide sequences longer than 30 residues; BLASTP using parameters of word length (W) of 2, expectation (E) of 1,000,000, and PAM30 scoring setting gap costs at 9 for open gaps and 1 for extended gaps for sequences shorter than 30 residues (default parameters for BLASTP are available at https: / / blast.ncbi.nlm.nih.gov); CLUSTALW using the Smith-Waterman homology search algorithm with parameters of match of 2, mismatch of -1, and gap of -1; MUSCLE using default parameters; MAFFT using parameters retree of 2 and maximum iterations of 1,000; Novafold using default parameters; and HMMER hmmalign using default parameters.

[0151] As used herein, the term "RuvC_III domain" refers to the third, non-contiguous segment of the RuvC endonuclease domain (the RuvC nuclease domain is composed of three non-contiguous segments, RuvC_I, RuvC_II, and RuvC_III). RuvC domains or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam HMM PF18541 for RuvC_III).

[0152] As used herein, the term "wedge" (WED) domain refers to a domain (e.g., present in Cas proteins) that primarily interacts with the repeat:anti-repeat duplex of the sgRNA and PAM duplex. WED domains can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences.

[0153] As used herein, the term "PAM-interacting domain" or "PI domain" refers to a domain that interacts with a protospacer adjacent motif (PAM) outside of the seed sequence within the region targeted by the Cas protein. Examples of PAM-interacting domains include, but are not limited to, the topoisomerase homology (TOPO) domain and the C-terminal domain (CTD) present in Cas proteins. PAM-interacting domains or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences.

[0154] As used herein, the term "REC domain" refers to a domain (e.g., present in a Cas protein) that contains at least one of two segments (REC1 or REC2) that are alpha helical domains thought to contact a guide RNA. REC domains or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam PF19501 for domain REC1).

[0155] As used herein, the term "BH domain" refers to a domain (e.g., present in a Cas protein) that is a bridging helix between the NUC and REC lobes of a Type II Cas enzyme. BH domains or segments thereof can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam PF16593 for domain BH).

[0156] As used herein, the term "HNH domain" refers to an endonuclease domain having characteristic histidine and asparagine residues. HNH domains can generally be identified by alignment to documented domain sequences, structural alignment to proteins with annotated domains, or comparison to hidden Markov models (HMMs) constructed based on documented domain sequences (e.g., Pfam HMM PF01844 for domain HNH).

[0157] Any variant of the enzymes described herein having one or more conservative amino acid substitutions is included in the present disclosure. Such conservative substitutions can be made in the amino acid sequence of the polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids with similar hydrophobicity, polarity, and R chain length. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by finding amino acid residues (e.g., non-conserved residues) that vary between species without altering the basic function of the encoded protein. Such conservatively substituted variants may have a similar or similar nucleotide sequence to any one of the systems described herein, such as 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%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at Conservatively substituted variants may include variants with at least about 3%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the endonuclease. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can include sequences with substitutions of key active site residues of the endonuclease such that the activity of the endonuclease is not disrupted. In some embodiments, functional variants of any of the systems described herein lack at least one substitution of a conserved or functional residue described herein. In some embodiments, functional variants of any of the systems described herein lack all substitutions of a conserved or functional residue described herein.

[0158] Conservative substitution tables providing functionally similar amino acids are available in various references (see, for example, Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman & Co.; 2nd Edition (December 1993))). The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), Threonine (T), and 8) Cysteine ​​(C), methionine (M).

[0159] overview The discovery of new Cas enzymes with unique functionality and structure could further disrupt deoxyribonucleic acid (DNA) editing technologies, offering the potential to improve speed, specificity, functionality, and ease of use. Compared to the predicted prevalence of clustered regularly interspaced short palindromic repeats (CRISPR) systems in microorganisms and the sheer diversity of microbial species, there are relatively few functionally characterized CRISPR / Cas enzymes in the literature. This is in part due to the inability to easily cultivate vast numbers of microbial species under laboratory conditions. Metagenomic sequencing from natural environmental niches representing numerous microbial species could dramatically increase the number of documented new CRISPR / Cas systems and potentially expedite the discovery of novel oligonucleotide editing functions. A recent example of the fruitfulness of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0160] CRISPR / Cas systems are RNA-directed nuclease complexes that have been described to function as adaptive immune systems in microorganisms. In their natural context, CRISPR / Cas systems occur in CRISPR (clustered regularly interspaced short palindromic repeats) operons or loci, which generally contain two parts: (i) an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences that encode RNA-based targeting elements; and (ii) an ORF encoding a Cas, which encodes a nuclease polypeptide directed by the RNA-based targeting element flanked by accessory proteins / enzymes. Efficient nuclease targeting of a specific target nucleic acid sequence can involve both (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide and (ii) the presence of a protospacer adjacent motif (PAM) sequence within a defined vicinity of the target seed (PAM is typically a sequence not commonly represented in the host genome). Depending on the exact function and organization of the system, CRISPR-Cas systems are generally organized into two classes, five types, and 16 subtypes based on shared functional characteristics and evolutionary similarities.

[0161] Class 1 CRISPR-Cas systems have large multi-subunit effector complexes and include Type I, Type III, and Type IV.

[0162] Type I CRISPR-Cas systems are considered to be of intermediate complexity in terms of components. In Type I CRISPR-Cas systems, an array of RNA targeting elements is transcribed as a long precursor crRNA (pre-crRNA) that is processed at the repeat element to release a short mature crRNA that directs the nuclease complex to the nucleic acid target, followed by a suitable short consensus sequence called a protospacer adjacent motif (PAM). This processing occurs via the endoribonuclease subunit (Cas6) of a larger endonuclease complex called Cascade, which also contains the nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nuclease functions primarily as a DNA nuclease.

[0163] Type III CRISPR systems can be characterized by the presence of a central nuclease known as Cas10, along with a repeat-associated mysterious protein (RAMP) containing Csm or Cmr protein subunits. Similar to Type I systems, mature crRNA is processed from pre-crRNA using a Cas6-like enzyme. Unlike Type I and II systems, Type III systems appear to target and cleave DNA-RNA duplexes (such as the DNA strand used as a template for RNA polymerase).

[0164] Type IV CRISPR-Cas systems possess an effector complex that includes a highly reduced large subunit nuclease (csf1), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) family, and in some cases, a predicted small subunit gene; such systems are typically found on endogenous plasmids.

[0165] Class 2 CRISPR-Cas systems generally have a single polypeptide multi-domain nuclease effector and include Type II, Type V, and Type VI.

[0166] Type II CRISPR-Cas systems are considered the simplest in terms of components. In Type II CRISPR-Cas systems, processing the CRISPR array into mature crRNA does not require the presence of a specialized endonuclease subunit, but rather a small transcoding crRNA (tracrRNA) with a region complementary to the array repeat sequence. The tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure, which is cleaved by endogenous RNAse III to generate the mature effector enzyme loaded with both the tracrRNA and crRNA. Cas II nucleases are documented as DNA nucleases. Type II effectors generally exhibit a structure containing a RuvC-like endonuclease domain that fits into an RNase H fold with an unrelated HNH nuclease domain inserted into the RuvC-like nuclease domain fold. The RuvC-like domain is responsible for cleavage of the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is responsible for cleavage of the displacement DNA strand.

[0167] Type V CRISPR-Cas systems are characterized by a nuclease effector (e.g., Cas12) structure similar to that of type II effectors, including a RuvC-like domain. Like type II, most (but not all) type V CRISPR systems use a tracrRNA to process pre-crRNA into mature crRNA. However, unlike type II systems, which require RNAse III to cleave the pre-crRNA into multiple crRNAs, type V systems can cleave the pre-crRNA using the effector nuclease itself. Like type II CRISPR-Cas systems, type V CRISPR-Cas systems are also documented as DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to possess robust single-strand nonspecific deoxyribonuclease activity that is activated by the first crRNA-directed cleavage of the double-stranded target sequence.

[0168] Type VI CRISPR-Cas systems have an RNA-guided RNA endonuclease. Instead of a RuvC-like domain, the single polypeptide effector of Type VI systems (e.g., Cas13) contains two HEPN ribonuclease domains. Unlike both Type II and V systems, Type VI systems also, in some embodiments, do not appear to require a tracrRNA to process pre-crRNA into crRNA. However, like Type V systems, some Type VI systems (e.g., C2C2) appear to have robust single-strand nonspecific nuclease (ribonuclease) activity that is activated by the first crRNA-directed cleavage of the target RNA.

[0169] Due to their simpler structure, Class 2 CRISPR-Cas have been most widely adopted for engineering and development as engineered nuclease / genome editing applications.

[0170] One of the initial adaptations of such a system for in vitro use involved (i) recombinantly expressed purified full-length Cas9 (e.g., Class 2, Type II Cas enzyme) isolated from S. pyogenes SF370, (ii) purified mature approximately 42-nt crRNA (total crRNA transcribed in vitro from a synthetic DNA template bearing a T7 promoter sequence) carrying an approximately 20-nt 5' sequence complementary to the target DNA sequence desired to be cleaved, followed by a 3' tracr binding sequence, (iii) purified tracrRNA transcribed in vitro from a synthetic DNA template bearing a T7 promoter sequence, and (iv) Mg 2+ Subsequent improved and engineered systems involved the crRNA (ii) joined to the 5' end of (iii) by a linker (e.g., GAAA) to form a single fusion synthetic guide RNA (sgRNA) that can itself guide Cas9 to the target.

[0171] Such engineered systems can be adapted for use in mammalian cells by providing a DNA vector encoding (i) an ORF encoding a codon-optimized Cas9 (e.g., a Class 2, Type II Cas enzyme) under a suitable mammalian promoter with a C-terminal nuclear localization sequence (e.g., SV40 NLS) and a suitable polyadenylation signal (e.g., a TK pA signal), and (ii) an ORF encoding an sgRNA (having a 5' sequence starting with G, followed by a 20 nt complementary targeting nucleic acid sequence attached to a 3' tracr binding sequence, a linker, and the tracrRNA sequence) under a suitable polymerase III promoter (e.g., a U6 promoter). Engineered endonucleases

[0172] In certain embodiments, engineered endonucleases are described herein. In some embodiments, the engineered endonucleases are chimeric of two or more endonucleases. In some embodiments, the engineered endonucleases are fusion of two or more endonucleases.

[0173] In some embodiments, the engineered endonuclease comprises one or more fragments or domains of a nuclease, such as a nucleic acid-guided nuclease. In some embodiments, the engineered endonuclease comprises one or more fragments or domains of a nuclease from an ortholog of an organism, genus, species, or other phylogenetic group described herein. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from a nuclease ortholog of a different species.

[0174] In some embodiments, the engineered endonuclease comprises one or more fragments or domains from at least two different nucleases. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleases. In some embodiments, the engineered endonuclease comprises one or more fragments or domains from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleases from different species. In some embodiments, the engineered endonuclease comprises two fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises three fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises four fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises five fragments or domains, each from a different nuclease. In some embodiments, the engineered endonuclease comprises three fragments or domains, and at least one fragment or domain is from a different nuclease. In some embodiments, the engineered endonuclease comprises four fragments or domains, and at least one fragment or domain is from a different nuclease. In some embodiments, the engineered endonuclease comprises five fragments or domains, and at least one fragment or domain is from a different nuclease.

[0175] In some embodiments, the engineered endonuclease is functional in prokaryotic or eukaryotic cells for in vitro, in vivo, or ex vivo use.

[0176] In some embodiments, the endonuclease is derived from an uncultivated microorganism. In some embodiments, the engineered endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease. In some embodiments, the engineered endonuclease has less than 86% identity to a SpyCas9 endonuclease.

[0177] In some embodiments, binding between fragments or domains from different nucleases or species occurs in stretches of unstructured regions. Unstructured regions in a polynucleotide include, for example, regions that lack predicted secondary structure elements, such as alpha helices or beta strands. Unstructured regions can include, for example, exposed regions in protein structures, loop regions, or regions that are not conserved among various protein orthologs, as predicted by sequence or structural alignment.

[0178] In certain embodiments, an N-terminal portion comprising 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 SEQ ID NO: 696 and at least 5% sequence identity to any one of SEQ ID NOs: 697-721. Described herein are engineered endonucleases (e.g., chimeric or fusion endonucleases) comprising an N-terminal portion comprising a sequence having at least about 70% identity to SEQ ID NO:696, and a C-terminal portion comprising a sequence having at least about 70% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 75% identity to SEQ ID NO: 696, and a C-terminal portion comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 80% identity to SEQ ID NO: 696, and a C-terminal portion comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 697-721.In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 85% identity to SEQ ID NO:696 and a C-terminal portion comprising a sequence having at least about 85% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 90% identity to SEQ ID NO:696 and a C-terminal portion comprising a sequence having at least about 90% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 95% identity to SEQ ID NO:696 and a C-terminal portion comprising a sequence having at least about 95% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 96% identity to SEQ ID NO:696, and a C-terminal portion comprising a sequence having at least about 96% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 97% identity to SEQ ID NO:696, and a C-terminal portion comprising a sequence having at least about 97% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 98% identity to SEQ ID NO:696, and a C-terminal portion comprising a sequence having at least about 98% identity to any one of SEQ ID NOs:697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having at least about 99% identity to SEQ ID NO: 696, and a C-terminal portion comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 697-721. In some embodiments, the engineered endonuclease comprises an N-terminal portion comprising a sequence having 100% identity to SEQ ID NO: 696, and a C-terminal portion comprising a sequence having 100% identity to any one of SEQ ID NOs: 697-721.

[0179] In certain embodiments, an N-terminal portion comprising the RuvC, REC, or HNH domain of a Cas endonuclease and comprising 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 SEQ ID NO: 696; and a WED, TOPO, or CTD domain of a Cas endonuclease. and a C-terminal portion comprising 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:697-721.

[0180] In some embodiments, the N-terminal portion of the engineered endonuclease comprises a RuvC domain, a REC domain, an HNH domain, a BH domain, or a combination thereof. In some embodiments, the C-terminal portion of the engineered endonuclease comprises a WED domain, a TOPO domain, a CTD domain, a PAM-interacting domain, or a combination thereof. In some embodiments, the N-terminal portion of the engineered endonuclease comprises a RuvC, REC, and HNH domain. In some embodiments, the N-terminal portion of the engineered endonuclease comprises a RuvC and an HNH domain. In some embodiments, the N-terminal portion of the engineered endonuclease further comprises a RuvC, REC, HNH, RuvC-I, BH, and RuvC-II domain. In some embodiments, the C-terminal portion of the engineered endonuclease comprises a WED, TOPO, and CTD domain. In some embodiments, the C-terminal portion of the engineered endonuclease comprises a PAM-interacting domain.

[0181] In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease do not occur together in the same reading frame in nature, hi some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are from different organisms.

[0182] In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are directly fused. In some embodiments, the N-terminal portion of the engineered endonuclease and the C-terminal portion of the engineered endonuclease are joined by a linker. In some embodiments, the linker is a glycine and / or serine-rich linker, a large protein domain, a long helix, or a short helix. In some embodiments, the linker is (GS)n, where n is an integer between 1 and 20. In some embodiments, the linker is GGGGS (SEQ ID NO: 2864). In some embodiments, the linker comprises a sequence selected from the group consisting of (GS)n, (GS)n, (GS)n, (GS)n, and (G)n, where n is an integer between 1 and 20. In some embodiments, one or more linkers comprise a sequence selected from the group consisting of (GGSGGD)n or (GGSGGE)n, where n is an integer between 1 and 6. In some embodiments, one or more linkers comprise a sequence selected from the group consisting of (GGGSGGG)n, (GGGSGSGGGGS)n, and (GGGGGPGGGGP)n, where n is an integer between 1 and 3. In some embodiments, one or more linkers comprise a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is between 1 and 20 and n is at least 8. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0183] In some embodiments, an engineered endonuclease (e.g., a chimeric endonuclease or a fusion endonuclease) 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: 1-27 and 771-862. In some embodiments, an engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862.In some embodiments, the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 1-27 and 771-862.

[0184] In some embodiments, the engineered endonucleases described herein have native PAM specificity (see FIG. 1B). In some embodiments, the present disclosure provides for achieving PAM specificity through protein engineering. In some embodiments, achieving PAM specificity is achieved by domain swapping of RNA-guided CRISPR-associated nucleases (see FIG. 2). In some embodiments, the domain swapping and recombination process has an optimal breakpoint. In some embodiments, the optimal breakpoint is guided by multiple sequence alignments as described herein (see FIG. 3A).

[0185] In some embodiments, the engineered endonuclease is configured to bind to a PAM that is not nnRGGnT. In some embodiments, the engineered endonuclease is configured to bind to a PAM in Table 1A ... In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of the following sequences: nnrmww, nnrRkyY, nnRnYhy, nnnmwTY, nnnmyY, nnRnYhY, nnnmhTY, nnRMAC, nnrRtwy, nnrwwYY, nnrmyY, nnrwYCC, nnRGnCr, nnnMhYy, nnyCCMww, nnnMCMCw, nnRwYhWw, nnRrYCYr, nnrwyhh, ntrMCm, nnrwyhh, nnRRTWY, ntTYCM, nnTCCC, nnrnyhh, nnnCCCYR, nnRnYAYn, nnRRnnYn, and nnRRnnYn. In some embodiments, the engineered endonuclease is configured to bind to the PAM by recognizing the PAM sequence. In some embodiments, the engineered endonuclease recognizes and binds to the PAM sequence.

[0186] [Table 1-1]

[0187] [Table 1-2]

[0188]

Table 1-3

[0189]

Table 1-4

[0190] In some embodiments, the engineered endonuclease (e.g., a chimeric endonuclease or a fusion endonuclease) 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: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 109-110 and 2842-2854.In some embodiments, the engineered endonuclease comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 109-110 and 2842-2854. In some embodiments, the engineered endonuclease comprises a sequence having 100% identity to any one of SEQ ID NOs: 109-110 and 2842-2854.

[0191] In some embodiments, the engineered endonuclease is configured to bind to a PAM that is not nnRGGnT. In some embodiments, the engineered endonuclease is configured to bind to a PAM in Table 1A. In some embodiments, the engineered endonuclease is configured to bind to a PAM comprising any one of TTTn, TYYn, TTTn, nYTn, TTYn, nTTn, nYYn, and nYYn. In some embodiments, the engineered endonuclease is configured to bind to a PAM by recognizing a PAM sequence. In some embodiments, the engineered endonuclease recognizes and binds to a PAM sequence.

[0192] Guide polynucleotide In certain embodiments, disclosed herein is an endonuclease system comprising: (a) an engineered nuclease disclosed herein; and (b) a guide polynucleotide, e.g., a guide ribonucleic acid (gRNA), a single gRNA, or dual guide RNAs. In polynucleotides, when referring to T, T means U (uracil) in RNA and T (thymine) in DNA.

[0193] In some embodiments, the engineered guide polynucleotide is configured to form a complex with the engineered endonuclease. In some cases, the engineered guide polynucleotide comprises a spacer sequence. In some cases, the spacer sequence is configured to hybridize to the target nucleic acid sequence. In some cases, the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence.

[0194] In some embodiments, the guide polynucleotide (e.g., gRNA) targets a gene or locus in a cell. In some embodiments, the guide polynucleotide targets a gene or locus in a mammalian cell. In some embodiments, the mammalian cell is a porcine, bovine, caprine, ovine, rodent, rat, mouse, non-human primate, or human cell. In some embodiments, the target gene or target locus is albumin, TRAC, B2M, TRBC1, TRBC2, ANGPTL3, PCSK9, VCP R155, AAVS1, GPR146, APOA1, or HAO1.

[0195] In some embodiments, the target gene is albumin. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 67-86, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 67-86.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 67-86.

[0196] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the albumin gene or an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 67-86, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 67-86.

[0197] In some embodiments, the target gene is TRAC. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.

[0198] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the TRAC gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to, or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to, any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.

[0199] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the TRAC gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.

[0200] In some embodiments, the target gene is B2M. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 159-184, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 159-184.

[0201] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the B2M gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 159-184, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 159-184.

[0202] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the B2M gene or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 185-210, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 185-210. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 185-210.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 185-210.

[0203] In some embodiments, the target gene is TRBC1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs:211-251, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:211-251. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs:211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs:211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 211-251.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 211-251.

[0204] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the TRBC1 gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 211-251, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 211-251.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 211-251.

[0205] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the TRBC1 gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 252-292, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 252-292. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 252-292.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 252-292.

[0206] In some embodiments, the target gene is TRBC2. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs:293-337, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs:293-337. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 293-337.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 293-337.

[0207] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the TRBC2 gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 293-337, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 293-337.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 293-337. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 293-337.

[0208] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the TRBC2 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 338-382, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 338-382. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 338-382.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 338-382.

[0209] In some embodiments, the target gene is ANGPTL3. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.

[0210] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the ANGPTL3 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to, or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to, any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.

[0211] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the ANGPTL3 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 478-572, 1490-1587, 2216-2311, and 2351-2389.

[0212] In some embodiments, the target gene is PCSK9. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 573-587 and 1362-1376, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.

[0213] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the PCSK9 gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 573-587 and 1362-1376, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 573-587 and 1362-1376. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 573-587 and 1362-1376.

[0214] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the PCSK9 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 588-602 and 1377-1391, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 588-602 and 1377-1391. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 588-602 and 1377-1391.

[0215] In some embodiments, the target gene is VCP (e.g., VCP R155). In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 723-738 and 755-762, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 723-738 and 755-762.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 723-738 and 755-762.

[0216] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the VCP gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 723-738 and 755-762, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 723-738 and 755-762.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 723-738 and 755-762. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 723-738 and 755-762.

[0217] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the VCP gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 739-754 and 763-770, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 739-754 and 763-770.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 739-754 and 763-770. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 739-754 and 763-770.

[0218] In some embodiments, the target locus is AAVS1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 928-949 and 1012-1049, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 928-949 and 1012-1049.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 928-949 and 1012-1049.

[0219] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the AAVS1 locus or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 928-949 and 1012-1049, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 928-949 and 1012-1049.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 928-949 and 1012-1049. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 928-949 and 1012-1049.

[0220] In some embodiments, the guide polynucleotide hybridizes or targets a sequence within the AAVS1 locus or within an intron of an endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence according to any one of SEQ ID NOs: 950-971 and 1050-1087, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 950-971 and 1050-1087.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 950-971 and 1050-1087. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 950-971 and 1050-1087.

[0221] In some embodiments, the target gene is GPR146. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 1588-1656, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1588-1656.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1588-1656.

[0222] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a target nucleic acid sequence within the GPR146 gene or an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 1588-1656, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1588-1656.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1588-1656. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 1588-1656.

[0223] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the GPR146 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 1657-1725, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1657-1725. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1657-1725.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 1657-1725.

[0224] In some embodiments, the target gene is APOA1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.

[0225] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the APOA1 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to, or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to, any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 80% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.

[0226] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence within the APOA1 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 80% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 85% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 90% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 95% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 96% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 97% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 98% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having at least about 99% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence having 100% identity to any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.

[0227] In some embodiments, the target gene is HAO1. In some embodiments, the guide polynucleotide is encoded by any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide comprises a sequence comprising at least about 46-80 contiguous nucleotides having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 80% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 85% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 90% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 95% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 96% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 97% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 98% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide is encoded by a sequence having 100% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.

[0228] In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to a target nucleic acid sequence within the HAO1 gene or within an intron of the endogenous gene. In some embodiments, the guide polynucleotide hybridizes or targets a sequence complementary to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. 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: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 85% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 90% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 95% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 96% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 97% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 98% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having at least about 99% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to a sequence having 100% identity to any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.

[0229] In some embodiments, the guide polynucleotide (e.g., guide RNA) comprises various structural elements, including, but not limited to, a spacer sequence that binds to a protospacer sequence (target sequence), a crRNA, and an optional tracrRNA. In some embodiments, the genome editing system comprises a CRISPR guide RNA. In some embodiments, the guide RNA comprises a crRNA that comprises a spacer sequence. In some embodiments, the guide RNA additionally comprises a tracrRNA or a modified tracrRNA.

[0230] In some embodiments, the systems provided herein include one or more guide RNAs. In some embodiments, the guide RNA includes a sense sequence. In some embodiments, the guide RNA includes an antisense sequence. In some embodiments, the guide RNA includes a nucleotide sequence other than a region complementary or substantially complementary to a region of the target sequence. For example, the crRNA is part of, or considered part of, the guide RNA, or is included in the guide RNA, e.g., a crRNA:tracrRNA chimera.

[0231] In some embodiments, the guide RNA comprises synthetic or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, the internucleoside linker of the guide RNA, or all of its contiguous nucleotide sequence, is modified. For example, in some embodiments, the internucleoside linkage comprises sulfur (S), such as a phosphorothioate internucleoside linkage. In some embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers. In some embodiments, the guide RNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 modified internucleoside linkers (e.g., phosphorothioate internucleoside linkages).

[0232] In some embodiments, the guide RNA comprises a modification to the ribose sugar or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides comprising a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribose nucleic acids (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, replacement with a hexose ring (HNA), a bicyclic ring having a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g., locked nucleic acids (LNA)), or an unlinked ribose ring that typically lacks a bond between the C2 and C3 carbons (e.g., UNA). In some embodiments, the sugar-modified nucleoside comprises a bicyclohexose nucleic acid or a tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as a peptide nucleic acid (PNA) or morpholino nucleic acid.

[0233] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, sugar modifications include modifications made by altering the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. In some embodiments, the substituent is introduced at the 2', 3', 4', 5' position, or a combination thereof. In some embodiments, nucleosides having modified sugar moieties include 2'-modified nucleosides, e.g., 2'-substituted nucleosides. 2'-sugar-modified nucleosides, in some embodiments, are nucleosides having a substituent other than H or -OH at the 2' position (2'-substituted nucleosides) or include a 2'-linked biradical, and include 2'-substituted nucleosides and LNA (2'-4' biradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification in the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0234] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In some embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methyl. In some embodiments, the modified sugar comprises a 2'-fluoro. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA 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).

[0235] In some embodiments, the guide RNA comprises both internucleoside linker modifications and nucleoside modifications. In some embodiments, the guide RNA comprises more than about 10%, 25%, 50%, 75%, or 90% modified internucleoside linkers and more than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the guide RNA 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).

[0236] In some cases, the guide RNA comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a eukaryotic genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a fungal genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a plant genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a mammalian genomic polynucleotide sequence. In some cases, the guide RNA comprises a sequence complementary to a human genomic polynucleotide sequence.

[0237] In some embodiments, the guide RNA is 30-250 nucleotides in length. In some embodiments, the guide RNA is more than 90 nucleotides in length. In some embodiments, the guide RNA is less than 245 nucleotides in length. In some embodiments, the guide RNA 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 RNAs are about 30 to about 40, about 30 to about 50, about 30 to about 60, about 30 to about 70, about 30 to about 80, about 30 to about 90, about 30 to about 100, about 30 to about 120, about 30 to about 140, about 30 to about 160, about 30 to about 180, about 30 to about 200, about 30 to about 220, about 30 to about 240, about 50 to about 60, about 50 to about 70, about 50 to about 80, about 50 to about 90, about 50 to about 100, about 50 The length is about 120, about 50 to about 140, about 50 to about 160, about 50 to about 180, about 50 to about 200, about 50 to about 220, about 50 to about 240, about 100 to about 120, about 100 to about 140, about 100 to about 160, about 100 to about 180, about 100 to about 200, about 100 to about 220, about 100 to about 240, about 160 to about 180, about 160 to about 200, about 160 to about 220, or about 160 to about 240 nucleotides.

[0238] MG endonuclease system 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 to hybridize to a target nucleic acid sequence.

[0239] In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide.In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide.

[0240] In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 70% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 75% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 80% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 85% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 90% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 95% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 96% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 97% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide.In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 98% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising a sequence having at least about 99% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide. In some embodiments, the engineered nuclease system comprises an engineered endonuclease comprising 100% identity to any one of SEQ ID NOs: 109-110 and 2842-2854, and an engineered guide polynucleotide.

[0241] In some embodiments, the engineered guide polynucleotide is a single guide nucleic acid. In some embodiments, the engineered guide polynucleotide is a dual guide nucleic acid. In some embodiments, the engineered guide polynucleotide is RNA. In some embodiments, the engineered endonuclease is non-covalently linked to the engineered guide polynucleotide. In some embodiments, the endonuclease is covalently linked to the engineered guide polynucleotide.

[0242] 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 67-86.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 67-86.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 67-86. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 67-86.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862, 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 albumin gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 67-86. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 67-86 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-86.

[0243] 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to, or having at least 90%, 95%, 97%, 98%, or 99% sequence identity to, any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.

[0244] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.

[0245] 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 159-184.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 159-184.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 159-184. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 159-184.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the engineered nuclease system comprises an endonuclease comprising 100% identity to any one of SEQ ID NOs: 1-27 and 771-862, and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises 100% identity to any one of SEQ ID NOs: 159-184. In some embodiments, the guide polynucleotide hybridizes to or targets a sequence complementary to any one of SEQ ID NOs: 159-184 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 159-184.

[0246] In some embodiments, the guide polynucleotide hybridizes to or targets a sequence according to any one of SEQ ID NOs: 185-210, or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 185-210. In some embodiments, the target nucleic acid sequence comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 185-210.

[0247] 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 211-251. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 211-251. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 211-251.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 211-251. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 211-251. 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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 211-251.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: 1-27 and 771-862; and an engineered guide polynucleotide configured to form a complex with the endonuclease and comprising a spacer sequence configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of the endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 211-251. In some embodiments, the engineered nuclease system comprises an endonuclease comprising a sequence having at...

Claims

1. A manipulated endonuclease, a) An N-terminal portion containing a sequence having at least 80% sequence identity with sequence number 696, b) A manipulated endonuclease comprising a C-terminal portion having at least 80% sequence identity with any one of sequence numbers 708, 697-707, and 709-721.

2. The manipulated endonuclease according to claim 1, wherein the N-terminal portion and the C-terminal portion are directly fused to each other or linked by a linker.

3. a) The linker is a glycine and / or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure, b) The linker is (GGGGS)n, where n is an integer from 1 to 20, and / or The manipulated endonuclease according to claim 2, wherein the linker is GGGGS (Sequence ID 2864).

4. The manipulated endonuclease according to claim 1, wherein the manipulated endonuclease is configured to bind to a PAM containing any one of sequence numbers 60-66, 117, 865-919, and 2855-2863.

5. An engineered endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27.

6. An engineered endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 779, 812, 814, 780, 811, and 817.

7. An engineered endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 109-110 and 2842-2854.

8. An engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) An engineered nuclease system comprising an engineered guide polynucleotide configured to form a complex with the engineered endonuclease and to hybridize to a target nucleic acid sequence.

9. a) The manipulated guide polynucleotide is a single guide nucleic acid, a dual guide nucleic acid, or RNA, and / or b) The manipulated nuclease system according to claim 8, wherein the manipulated endonuclease is non-covalently bonded to the manipulated guide polynucleotide, or the manipulated endonuclease is covalently bonded to the manipulated guide polynucleotide, or the manipulated endonuclease is fused to the manipulated guide polynucleotide.

10. The manipulated nuclease system according to claim 8, wherein the manipulated guide polynucleotide comprises a sequence having at least 90% or 100% sequence identity with any one of SEQ ID NOs. 28-45, 605-610, 646-695, 863, and 1789-1826.

11. The manipulated nuclease system according to claim 8, wherein the manipulated endonuclease is configured to bind to a PAM containing any one of sequence numbers 60-66, 865-919, and 2863.

12. An engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) An engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within an albumin gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 67 to 86.

13. An engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) An engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within a TRAC gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.

14. The manipulated nuclease system according to claim 13, wherein the target nucleic acid sequence includes a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 139-158, 925-927, 992-1011, 1184-1279, 1321-1361, 2486-2581, and 2618-2653.

15. An engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) (i) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 159 to 184, (ii) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 211 to 251, (iii) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the TRBC2 gene or intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 293 to 337, (iv) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350. (v) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the PCSK9 gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 573-587 and 1362-1376, (vi) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within a VCP gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 723-738 and 755-762, (vii) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the AAVS1 locus or intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 928-949 and 1012-1049, (viiii) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the GPR146 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 1588 to 1656, (ix) An engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the APOA1 gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 1726-1744, 1764-1774, 1866-1961, and 2058-2088, or (x) An engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the HAO1 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 611-633, 1789-1826, and 1827-1865.

16. a) The target nucleic acid sequence is located within the B2M gene or within the intron of an endogenous gene and contains a sequence having at least 90% sequence identity with any one of sequence numbers 185 to 210, b) The target nucleic acid sequence is located within the TRBC1 gene or within an intron of an endogenous gene and contains a sequence that has at least 90% sequence identity with any one of sequence numbers 252 to 292, c) The target nucleic acid sequence is located within the TRBC2 gene or within the intron of an endogenous gene and contains a sequence that has at least 90% sequence identity with any one of sequence numbers 338 to 382, d) The target nucleic acid sequence is located within the ANGPTL3 gene or within the intron of an endogenous gene and contains a sequence that has at least 90% sequence identity with any one of sequence numbers 478-572, 1490-1587, 2216-2311, and 2351-2389, e) The target nucleic acid sequence is located within the PCSK9 gene or within the intron of an endogenous gene and contains a sequence having at least 90% sequence identity with any one of sequence numbers 588-602 and 1377-1391, f) The target nucleic acid sequence is located within the VCP gene or within the intron of an endogenous gene and contains a sequence that has at least 90% sequence identity with any one of sequence numbers 739-754 and 763-770, g) The target nucleic acid sequence is located within the AAVS1 gene locus or within the intron of an endogenous gene and contains a sequence having at least 90% sequence identity with any one of sequence numbers 950-971 and 1050-1087, h) The target nucleic acid sequence is located within the GPR146 gene or within an intron of an endogenous gene and contains a sequence that has at least 90% sequence identity with any one of sequence numbers 1657 to 1725, i) The manipulated nuclease system according to claim 15, wherein the target nucleic acid sequence is located within the APOA1 gene or within an intron of an endogenous gene and has at least 90% sequence identity with any one of SEQ ID NOs: 1745-1763, 1775-1785, 1962-2057, and 2089-2119.

17. A method for modifying a target nucleic acid sequence, comprising contacting the target nucleic acid sequence with an engineered endonuclease according to any one of claims 1 to 7 or an engineered nuclease system according to any one of claims 8 to 16.

18. The method according to claim 17, wherein the target nucleic acid sequence includes a sequence having any one of sequence numbers 261-270, 275-278, and 291-302.

19. Use of an engineered endonuclease according to any one of claims 1 to 7 or an engineered nuclease system according to any one of claims 8 to 16 for modifying a target nucleic acid sequence in a mammalian cell, the use comprising contact with the mammalian cell.

20. A method for modifying an albumin gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the albumin gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the albumin gene or an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 67 to 86.

21. A method for modifying the TRAC gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the TRAC gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the TRAC gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 119-138, 922-924, 972-991, 1088-1183, 1280-1320, 2390-2485, and 2582-2617.

22. A method for modifying the B2M gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the B2M gene using an engineered nuclease system, the engineered guide polynucleotide comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the B2M gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 159 to 184.

23. A method for modifying the TRBC1 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the TRBC1 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC1 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 211 to 251.

24. A method for modifying the TRBC2 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the TRBC2 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the TRBC2 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 293 to 337.

25. A method for modifying the ANGPTL3 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the ANGPTL3 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the ANGPTL3 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 383-477, 1392-1489, 2120-2215, and 2312-2350.

26. A method for modifying the PCSK9 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the PCSK9 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the PCSK9 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 573-587 and 1362-1376.

27. ​​A method for modifying the VCP gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the VCP gene using an engineered nuclease system, the engineered guide polynucleotide comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the VCP gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 723-738 and 755-762.

28. A method for modifying the AAVS1 gene locus, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the AAVS1 locus using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the AAVS1 locus or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs. 928-949 and 1012-1049.

29. A method for modifying the GPR146 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the GPR146 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the GPR146 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of sequence numbers 1588 to 1656.

30. A method for modifying the APOA1 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the APOA1 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and configured to hybridize to at least a portion of a target nucleic acid sequence within the APOA1 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 1726-1744, 1764-1774, 1866-1961, and 2058-2088.

31. A method for modifying the HAO1 gene, wherein the method comprises an engineered nuclease system, a) A manipulated endonuclease comprising a sequence having at least 80% sequence identity with any one of sequence numbers 12, 773, 771-772, 774-862, 1-11, and 13-27, b) A method comprising contacting the HAO1 gene using an engineered nuclease system comprising an engineered guide polynucleotide comprising a spacer sequence configured to form a complex with the engineered endonuclease and to hybridize to at least a portion of a target nucleic acid sequence within the HAO1 gene or within an intron of an endogenous gene, wherein the engineered guide polynucleotide comprises a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 611-633, 1789-1826, and 1827-1865.

32. A cell comprising an engineered endonuclease according to any one of claims 1 to 7, or an engineered nuclease system according to any one of claims 8 to 16.

33. The cell is a) eukaryotic cells, b) mammalian cells; c) immortalized cells; d) insect cells; e) yeast cells, f) plant cells; g) fungal cells; h) prokaryotic cells; i) A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or their derivatives, ms) Manipulated cells, or k) The cell according to claim 32, which is a stable cell.