Enzymes containing RUVC domains

Abstract

The present disclosure provides endonuclease enzymes with distinct domain characteristics, as well as methods of using such enzymes or variants thereof.

Description

[Background technology]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 182,438, entitled "ENZYMES WITH RUVC DOMAINS," filed April 30, 2021, which is incorporated by reference in its entirety.

[0002] Cas enzymes, along with their associated clustered regularly interspaced short palindromic repeats (CRISPR)-guided ribonucleic acid (RNA), appear to be widespread components of the immune system of prokaryotes (~45% of bacteria, ~84% of archaea), helping to protect these microorganisms from non-self nucleic acids such as infectious viruses and plasmids by 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. Although CRISPR DNA elements have been 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 engineering and gene editing applications. Summary of the Invention

[0003] In some aspects, the disclosure provides an engineered nuclease system comprising: (a) an endonuclease configured to bind to a protospacer adjacent motif (PAM) sequence comprising SEQ ID NOs: 550-567, the endonuclease being a class 2 type II Cas endonuclease; and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease comprising (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) 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 engineered to bind to a different PAM sequence. 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 80% identity to a Cas9 endonuclease. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides. In some embodiments, the engineered guide ribonucleic acid structure comprises a ribonucleic acid polynucleotide comprising the guide ribonucleic acid sequence and the tracr ribonucleic acid sequence. In some embodiments, the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the guide ribonucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises a sequence selected from SEQ ID NOs: 586-601.In some embodiments, the engineered nuclease system further comprises a single-stranded or double-stranded DNA repair template comprising, in a 5' to 3' direction, a first homologous arm comprising a sequence of at least 20 nucleotides 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homologous arm comprising a sequence of at least 20 nucleotides 3' to the target sequence. In some embodiments, the first or second homologous arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. In some embodiments, the system comprises Mg. 2+ In some embodiments, the endonuclease and the tracr ribonucleic acid sequence are derived from distinct bacterial species within the same phylum. In some embodiments, the endonuclease comprises SEQ ID NO: 1-549 or 602-1276, or a variant thereof having at least 55% identity thereto.

[0004] In one aspect, the disclosure provides an engineered nuclease system comprising (a) an endonuclease configured to have selectivity for a protospacer adjacent motif (PAM) sequence comprising any one of SEQ ID NOs: 550-567, wherein the endonuclease is a class 2 type II Cas endonuclease. In some embodiments, the system further comprises (b) an engineered guide nucleic acid structure configured to form a complex with the endonuclease, the guide nucleic acid structure comprising (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) 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 engineered to bind to a PAM sequence that is different from the native PAM sequence of the endonuclease. 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 80% identity with a Cas9 endonuclease. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the HNH domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease is configured to have selectivity for a PAM sequence comprising any one of SEQ ID NOs: 553, 555, or 566, or a variant thereof. In some embodiments, the engineered guide nucleic acid structure comprises at least two ribonucleic acid polynucleotides. In some embodiments, the engineered guide nucleic acid structure comprises one ribonucleic acid polynucleotide comprising a guide ribonucleic acid sequence and a tracr ribonucleic acid sequence. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or an engineered The constructed guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The guide nucleic acid structure, which may be comprised or engineered, comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the targeting nucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. The NLS comprises a sequence comprising any one of SEQ ID NOs: 586-601, or a variant thereof. In some embodiments, the system further comprises a single-stranded or double-stranded DNA repair template comprising, in a 5' to 3' direction, a first homologous arm comprising a sequence of at least 20 nucleotides 5' to a target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homologous arm comprising a sequence of at least 20 nucleotides 3' to the target sequence. In some embodiments, the first or second homologous arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. In some embodiments, the system further comprises a Mg. 2+In some embodiments, the endonuclease and the tracr ribonucleic acid sequence are derived from distinct bacterial species within the same phylum. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity thereto. In some embodiments, the sequence identity is determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, or CLUSTALW algorithm with Smith-Waterman homology search algorithm parameters. In some embodiments, the sequence identity is determined by the BLASTP homology search algorithm using the BLOSUM62 scoring matrix with parameters of word length (W) of 3, expectation (E) of 10, and gap cost set at 11 presence, 1 extension, and with a conditional composition score matrix adjustment. In some embodiments, the PAM sequence is 3' to the target deoxyribonucleic acid sequence.

[0005] In some aspects, the disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, the nucleic acid encoding a Class 2 Type II Cas endonuclease configured to be selective for a protospacer adjacent motif (PAM) comprising any one of SEQ ID NOs: 550-567. In some embodiments, the endonuclease comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain.In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the organism is a bacterium, an archaea, a eukaryote, a fungus, a plant, a mammal, or a human organism.

[0006] In some aspects, the disclosure provides an engineered nuclease system comprising: (a) an endonuclease having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1-549, 602-1276, or a variant thereof; and (b) an engineered guide nucleic acid structure, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting nucleic acid sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and comprises (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.

[0007] In some embodiments, the disclosure provides a method for the preparation of a tracr sequence having (a) (i) at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662; or (ii) a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 568-585 or 1643-16 44 non-degenerate nucleotides of at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (b) a Class 2 Type II Cas endonuclease configured to bind to the engineered guide nucleic acid structure. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 571, 573, or 584 non-degenerate nucleotides. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and includes (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.In some embodiments, the endonuclease comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1-549, 602-1276, or a variant thereof. In some embodiments, the endonuclease comprises a sequence set forth in any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0008] In some aspects, the disclosure provides engineered guide nucleic acid structures comprising: (a) a targeting nucleic acid sequence comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule; and (b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex, one of which comprises a tracr sequence, wherein the two complementary stretches of nucleotides are covalently linked to each other with an intervening nucleotide, wherein the engineered guide ribonucleic acid polynucleotide is selected from the group consisting of SEQ ID NOs: 1-549, 602-603, 604-605, 606-607, 608-609, 610-611, 612-613, 614-615, 616-617, 618-619, 620-621, 622-623, 624-625, 626-627, 628-629, 630-631, 632-633, 634-635, 636-637, 638-639, 640-641, 642-643, 644-645, 646-647, 648-650, 649-651, 652-653, 654-655, 656-657, 658-659, 660-661, 661-670, 662-671, 663-672, 664-673, 665-674, 666-675, 676-677, 678-679, 679-700, 679-701, 1276, or a variant thereof, and capable of forming a complex with an endonuclease having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the endonuclease and targeting the complex to a target sequence of a target DNA molecule. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584.

[0009] In some aspects, the present disclosure provides an engineered vector comprising any of the nucleic acids described herein. In some embodiments, the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV)-derived virion, a lentivirus, or an adenovirus.

[0010] In some aspects, the present disclosure provides a cell comprising any of the vectors described herein or any of the nucleic acids described herein. In some embodiments, the cell is a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human cell.

[0011] In some aspects, the disclosure provides a method of producing an endonuclease comprising culturing any of the cells described herein.

[0012] In some aspects, the disclosure provides a method of binding, cleaving, marking or modifying a double-stranded deoxyribonucleic acid polynucleotide comprising contacting the double-stranded deoxyribonucleic acid polynucleotide with a Class 2 Type II Cas endonuclease in a complex with an endonuclease and an engineered guide nucleic acid structure configured to bind to the double-stranded deoxyribonucleic acid polynucleotide, wherein the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM), and the PAM comprises a sequence set forth in any one of SEQ ID NOs: 550-567. In some embodiments, the endonuclease comprises a PI domain that includes a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof.In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or an engineered The constructed guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and comprises (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.

[0013] In some aspects, the disclosure provides a method of editing the AAVS1 locus in a cell, comprising contacting the cell with: (a) an RNA-guided endonuclease; and (b) an engineered guide nucleic acid structure, wherein the engineered guide nucleic acid structure is configured to form a complex with the endonuclease; and wherein the engineered guide nucleic acid structure comprises a spacer sequence configured to hybridize to a region of the AAVS1 locus, wherein the engineered guide nucleic acid structure comprises a targeting sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least 18 contiguous nucleotides of any one of SEQ ID NOs: 1665-1666, or its reverse complement. In some embodiments, the engineered guide nucleic acid structure has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1663 or 1664. In some embodiments, the engineered guide nucleic acid structure is MG71-2-AAVS1-sgRNA-C3 or MG71-2-AAVS1-sgRNA-E2.

[0014] In some aspects, the disclosure provides a method of editing a TRAC locus in a cell, comprising contacting the cell with: (a) an RNA-guided endonuclease; and (b) an engineered guide nucleic acid structure, wherein the engineered guide nucleic acid structure is configured to form a complex with the endonuclease; and wherein the engineered guide nucleic acid structure comprises a spacer sequence configured to hybridize to a region of the TRAC locus, and wherein the engineered guide nucleic acid structure comprises a targeting sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least 18 contiguous nucleotides of any one of SEQ ID NOs: 1668 or 1676-1682, or its reverse complement. In some embodiments, the engineered guide nucleic acid structure has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1667 or 1669-1675. In some embodiments, the engineered guide nucleic acid structure is MG73-1-TRAC-sgRNA-G3, MG89-2-TRAC-sgRNA-F1, MG89-2-TRAC-sgRNA-G5, MG89-2-TRAC-sgRNA-E5, MG89-2-TRAC-sgRNA-F5, MG89-2-TRAC-sgRNA-G1, MG89-2-TRAC-sgRNA-E1, MG89-2-TRAC-sgRNA-B1. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and includes (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 571, 573, or 584. In some embodiments, the RNA-guided endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the endonuclease is configured to have a preference for PAM sequences comprising any one of SEQ ID NOs: 550-567.In some embodiments, the endonuclease comprises a PI domain that includes a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0015] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details can be modified 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.

[0016] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0017] 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 invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "FIG" and "FIG.") [Figure 1] FIG. 1 shows typical organizations of different classes and types of CRISPR / Cas loci. [Diagram 2] FIG. 2 shows the structure of the natural class II / type II crRNA / tracrRNA pair compared to a hybrid sgRNA to which both are bound. [Figure 3A] FIG. 3 (e.g., FIGS. 3A, 3B, 3C, 3D, and 3E) shows seqLogo representations of PAM sequences derived via NGS as described herein (e.g., as described in Example 3). [Figure 3B] FIG. 3 (e.g., FIGS. 3A, 3B, 3C, 3D, and 3E) shows seqLogo representations of PAM sequences derived via NGS as described herein (e.g., as described in Example 3). [Figure 3C] FIG. 3 (e.g., FIGS. 3A, 3B, 3C, 3D, and 3E) shows seqLogo representations of PAM sequences derived via NGS as described herein (e.g., as described in Example 3). [Figure 3D] FIG. 3 (e.g., FIGS. 3A, 3B, 3C, 3D, and 3E) shows seqLogo representations of PAM sequences derived via NGS as described herein (e.g., as described in Example 3). [Figure 3E] FIG. 3 (e.g., FIGS. 3A, 3B, 3C, 3D, and 3E) shows seqLogo representations of PAM sequences derived via NGS as described herein (e.g., as described in Example 3). [Figure 4] FIG. 4 shows the results of gene editing at the DNA level for TRAC and AAVS1 in K562 cells in Example 7.

[0018] 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.

[0019] MG44

[0020] SEQ ID NOs: 1 to 216 and 602 to 938 show the full-length peptide sequences of MG44 nuclease.

[0021] SEQ ID NO:550 shows a PAM sequence compatible with MG44 nuclease.

[0022] SEQ ID NO: 568 shows the nucleotide sequence of an sgRNA engineered to function with MG44 nuclease, where Ns shows the nucleotide of the targeting sequence.

[0023] SEQ ID NOs: 1277 to 1419 show the peptide sequences of the PAM interaction domain of MG44 nuclease.

[0024] SEQ ID NO: 1645 shows the nucleotide sequence of MG44 tracrRNA, which is derived from the same locus as the MG44 nuclease described above.

[0025] MG46

[0026] SEQ ID NOs: 217 to 257 and 939 to 1104 show the full-length peptide sequences of MG46 nuclease.

[0027] SEQ ID NO:551 shows a PAM sequence compatible with MG46 nuclease.

[0028] SEQ ID NO: 569 shows the nucleotide sequence of an sgRNA engineered to function with MG46 nuclease, where Ns shows the nucleotide of the targeting sequence.

[0029] SEQ ID NOs: 1420 to 1497 show the peptide sequences of the PAM interaction domain of MG46 nuclease.

[0030] SEQ ID NO: 1646 shows the nucleotide sequence of the MG46 tracrRNA, which is derived from the same locus as the MG46 nuclease described above.

[0031] MG71

[0032] SEQ ID NOs: 258 to 283 and 1105 show the full-length peptide sequences of MG71 nuclease.

[0033] SEQ ID NOs: 552 to 553 show PAM sequences compatible with MG71 nuclease.

[0034] SEQ ID NOs: 570-571 show the nucleotide sequences of sgRNAs engineered to function with MG71 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0035] SEQ ID NOs: 1498 to 1499 show the peptide sequences of the PAM interaction domain of MG71 nuclease.

[0036] SEQ ID NOs: 1643-1644 show the nucleotide sequences of sgRNAs engineered to function with MG71 nuclease.

[0037] SEQ ID NOs: 1647-1648 show the nucleotide sequence of MG71 tracrRNA, which is derived from the same locus as the MG71 nuclease described above.

[0038] MG72

[0039] SEQ ID NOs: 284 to 295 and 1106 to 1115 show the full-length peptide sequences of MG72 nuclease.

[0040] SEQ ID NO:554 shows a PAM sequence compatible with MG72 nuclease.

[0041] SEQ ID NO: 572 shows the nucleotide sequence of an sgRNA engineered to function with MG72 nuclease, where Ns shows the nucleotide of the targeting sequence.

[0042] SEQ ID NO: 1649 shows the nucleotide sequence of the MG72 tracrRNA, which is derived from the same locus as the MG72 nuclease described above.

[0043] MG73

[0044] SEQ ID NOs: 296 to 305 and 1116 to 1118 show the full-length peptide sequences of MG73 nuclease.

[0045] SEQ ID NO:555 shows a PAM sequence compatible with MG73 nuclease.

[0046] SEQ ID NOs: 573-574 show the nucleotide sequences of sgRNAs engineered to function with MG73 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0047] SEQ ID NOs: 1500 to 1505 show the peptide sequences of the PAM interaction domain of MG73 nuclease.

[0048] SEQ ID NOs: 1650-1651 show the nucleotide sequence of MG73 tracrRNA, which is derived from the same locus as the MG73 nuclease described above.

[0049] MG74

[0050] SEQ ID NOs: 306 to 355 and 1119 to 1160 show the full-length peptide sequences of MG74 nuclease.

[0051] SEQ ID NO:556 shows a PAM sequence compatible with MG74 nuclease.

[0052] SEQ ID NO: 575 shows the nucleotide sequence of an sgRNA engineered to function with MG74 nuclease, where Ns shows the nucleotide of the targeting sequence.

[0053] SEQ ID NOs: 1506 to 1519 show the peptide sequences of the PAM interaction domain of MG74 nuclease.

[0054] SEQ ID NO: 1652 shows the nucleotide sequence of the MG74 tracrRNA, which is derived from the same locus as the MG74 nuclease described above.

[0055] MG86

[0056] SEQ ID NOs: 356 to 402 and 1161 to 1206 show the full-length peptide sequences of MG86 nuclease.

[0057] SEQ ID NOs: 557 to 559 show PAM sequences compatible with MG86 nuclease.

[0058] SEQ ID NOs: 576-577 show the nucleotide sequences of sgRNAs engineered to function with MG86 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0059] SEQ ID NOs: 1520 to 1578 show the peptide sequences of the PAM interaction domain of MG86 nuclease.

[0060] SEQ ID NO: 1642 shows the nucleotide sequence of the single guide PAM of MG86 nuclease.

[0061] SEQ ID NOs: 1653-1654 show the nucleotide sequence of MG86 tracrRNA, which is derived from the same locus as the MG86 nuclease described above.

[0062] MG87

[0063] SEQ ID NOs: 403 to 462 and 1207 to 1247 show the full-length peptide sequences of MG87 nuclease.

[0064] SEQ ID NOs:560 to 562 show PAM sequences compatible with MG87 nuclease.

[0065] SEQ ID NOs: 578-580 show the nucleotide sequences of sgRNAs engineered to function with MG87 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0066] SEQ ID NOs: 1579 to 1615 show the peptide sequences of the PAM interaction domain of MG87 nuclease.

[0067] SEQ ID NOs: 1655-1657 show the nucleotide sequence of MG87 tracrRNA, which is derived from the same locus as the MG87 nuclease described above.

[0068] MG88

[0069] SEQ ID NOs: 463 to 482 and 1248 to 1258 show the full-length peptide sequences of MG88 nuclease.

[0070] SEQ ID NOs:563 to 565 show PAM sequences compatible with MG88 nuclease.

[0071] SEQ ID NOs: 581-583 show the nucleotide sequences of sgRNAs engineered to function with MG88 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0072] SEQ ID NOs: 1616 to 1628 show the peptide sequences of the PAM interaction domain of MG88 nuclease.

[0073] SEQ ID NOs: 1658-1660 show the nucleotide sequence of MG88 tracrRNA, which is derived from the same locus as the MG88 nuclease described above.

[0074] MG89

[0075] SEQ ID NOs: 483 to 549 and 1259 to 1276 show the full-length peptide sequences of MG89 nuclease.

[0076] SEQ ID NOs:566-567 show PAM sequences compatible with MG89 nuclease.

[0077] SEQ ID NOs: 584-585 show the nucleotide sequences of sgRNAs engineered to function with MG89 nuclease, where Ns shows the nucleotides of the targeting sequence.

[0078] SEQ ID NOs: 1629 to 1641 show the peptide sequences of the PAM interaction domain of MG89 nuclease.

[0079] SEQ ID NOs: 1661-1662 show the nucleotide sequence of MG88 tracrRNA, which is derived from the same locus as the MG88 nuclease described above.

[0080] MG71-2 AAVS1 targeting SEQ ID NOs: 1663-1664 show the nucleotide sequences of sgRNAs engineered to function with MG71-2 nuclease to target AAVS1.

[0081] SEQ ID NOs: 1665 to 1666 show the DNA sequence of the AAVS1 target site.

[0082] MG73-1 TRAC targeting SEQ ID NO: 1667 shows the nucleotide sequence of an sgRNA engineered to function with MG73-1 nuclease to target TRAC.

[0083] SEQ ID NO: 1668 shows the DNA sequence of the TRAC target site.

[0084] MG89-2 TRAC targeting SEQ ID NOs: 1669-1675 show the nucleotide sequences of sgRNAs engineered to function with MG89-2 nuclease to target TRAC.

[0085] SEQ ID NOs: 1676 to 1682 show the DNA sequences of the TRAC target sites. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] While various embodiments of the present invention 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 invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0087] The practice of some methods disclosed herein, unless otherwise indicated, uses immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA techniques.See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMA Usubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)) (fully incorporated herein by reference).

[0088] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent the terms "comprising," "including," "having," "having," "having," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0089] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0090] As used herein, "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-cell 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, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, hornworts, bryophytes, mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. agarwal), and the like). C. Agardh), 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 and sometimes referred to as artificial cells).

[0091] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. A nucleotide may include synthetic nucleotides. A nucleotide may include synthetic nucleotide analogs. A nucleotide may be a monomeric unit of a nucleic acid sequence (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 their derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled, such as by using a moiety that includes an optically detectable moiety (e.g., a fluorophore). Labeling may also be performed using quantum dots. Detectable labels may 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.; fluoro-conjugated deoxynucleotides, fluoro-conjugated Cy3-dCTP, fluoro-conjugated Cy5-dCTP, fluoro-conjugated fluoroX-dCTP, fluoro-conjugated Cy3-dUTP, and fluoro-conjugated Cy5-dUTP available from Amersham, Arlington Heights, Ill.; 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 labeling 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 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).

[0092] The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are generally used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-stranded, double-stranded, 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. A polynucleotide 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 the 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 a gene or gene fragment, loci defined from binding analyses, 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.

[0093] The term "transfection" or "transfected" generally refers to the introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecule may be a genetic sequence encoding a complete protein or a functional portion thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0094] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues linked by a peptide bond(s). The term does not refer to 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 containing 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 (e.g., domains) with or without secondary and / or tertiary structure. The term also encompasses amino acid polymers modified by any other manipulation, such as, for example, 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" generally refer to natural and non-natural 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 a non-naturally occurring group or chemical moiety on the amino acid. An amino acid analog may refer to an amino acid derivative. The term "amino acid" includes both D- and L-amino acids.

[0095] As used herein, "non-natural" can generally refer to a nucleic acid or polypeptide sequence that is not present in a naturally occurring nucleic acid or protein. Non-natural can refer to an affinity tag. Non-natural can refer to a fusion. Non-natural can refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-natural sequence can exhibit and / or encode an activity (e.g., an enzyme activity, a methyltransferase activity, an acetyltransferase activity, a kinase activity, an ubiquitination activity, etc.) that can also be exhibited by the nucleic acid sequence and / or polypeptide sequence to which the non-natural sequence is fused. A non-natural nucleic acid or polypeptide sequence can be linked to a naturally occurring nucleic acid and / or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and / or polypeptide sequence that encodes a chimeric nucleic acid or polypeptide.

[0096] As used herein, the term "promoter" generally refers to a regulatory DNA region that controls the transcription or expression of a gene and may be located adjacent to or overlapping the nucleotide or region of nucleotides 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 called a "core promoter", may generally refer to a promoter that contains all the basic essential 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.

[0097] As used herein, the term "expression" generally 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) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in eukaryotic cells.

[0098] As used herein, "operably linked," "operably linked," "operably linked," or grammatical equivalents thereof generally refer to the juxtaposition of genetic elements, such as promoters, enhancers, polyadenylation sequences, and the like, where the elements are in a relationship that allows them to operate in an expected manner. 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 initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

[0099] As used herein, a "vector" generally refers to a polymer or an association of polymers that contains or associates 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 includes a genetic element, such as a control element, that is operably linked to a gene to facilitate expression of the gene in a target.

[0100] As used herein, "expression cassette" and "nucleic acid cassette" are generally used interchangeably to refer to a nucleic acid sequence or combination of elements 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.

[0101] A "functional fragment" of a DNA or protein sequence generally 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 known to be attributable to the full-length sequence.

[0102] As used herein, an "engineered" subject generally indicates that the subject has been modified by human intervention. By way of non-limiting examples, a nucleic acid may be modified by altering its sequence to a sequence that does not occur in nature, a nucleic acid may be modified by ligating to a nucleic acid with which it is not naturally associated such that the ligated product has a function not present in the original nucleic acid, an engineered nucleic acid may be synthesized in vitro with a sequence that does not occur in nature, a protein may be modified by changing its amino acid sequence to a sequence that does not occur in nature, and an engineered protein may acquire a new function or property. An "engineered" system includes at least one engineered component.

[0103] As used herein, "synthetic" and "artificial" are used interchangeably to refer to proteins or domains thereof that 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) with naturally occurring human proteins. For example, the VPR domain and the VP64 domain are synthetic transactivation domains.

[0104] As used herein, the term "tracrRNA" or "tracr sequence" can generally refer to a nucleic acid having at least 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 Streptococcus pyogenes, Staphylococcus aureus, etc.). A tracrRNA can refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.). tracrRNA refers to modified forms of tracrRNA that may include nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeras. tracrRNA may refer to a nucleic acid that may be at least about 60% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus) sequence over a stretch of at least six consecutive 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., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least six consecutive nucleotides. Type II tracrRNA sequences can be predicted on a genomic sequence by identifying regions that have complementarity to parts of the repeat sequences in the adjacent CRISPR array.

[0105] As used herein, a "guide nucleic acid" can generally refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed to bind to a sequence of a nucleic acid in a site-specific manner. The nucleic acid to be targeted, or the target nucleic acid, can include nucleotides. A guide nucleic acid can include nucleotides. A portion of a target nucleic acid can be complementary to a portion of a guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to a guide nucleic acid and hybridizes with the guide nucleic acid can be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore not complementary to the guide nucleic acid can be referred to as a non-complementary strand. A guide nucleic acid can include a polynucleotide strand and can be referred to as a "single guide nucleic acid". A guide nucleic acid can include two polynucleotide strands and can be referred to as a "double guide nucleic acid". Otherwise, the term "guide nucleic acid" can be inclusive, 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 a "nucleic acid targeting sequence." The nucleic acid targeting segment may include a sub-segment that may be referred to as a "protein binding segment" or a "protein binding sequence" or a "Cas protein binding segment."

[0106] The term "sequence identity" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences generally refers to two (e.g., in a pairwise alignment) or more (e.g., in a multiple sequence alignment) sequences that are identical or have a certain 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 an 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 in BLAST 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 with default parameters; MAFFT with parameters retree of 2 and maximum iterations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

[0107] As used herein, the term "RuvC_III domain" generally refers to the third discontinuous segment of the RuvC endonuclease domain (the RuvC nuclease domain is composed of three discontinuous segments, RuvC_I, RuvC_II, and RuvC_III). The RuvC domain or a segment thereof can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or comparison to Hidden Markov Models (HMMs) constructed based on known domain sequences (e.g., Pfam HMM PF18541 for RuvC_III).

[0108] As used herein, the term "HNH domain" generally refers to the endonuclease domain that has characteristic histidine and asparagine residues.HNH domain can generally be identified by alignment to known domain sequences, structural alignment to proteins with annotated domains, or comparison with Hidden Markov Models (HMM) that are constructed based on known domain sequences (e.g., Pfam HMM PF01844 of domain HNH).

[0109] As used herein, the term "wedge" (WED) domain generally refers to a domain (e.g., present in Cas proteins) that primarily interacts with the sgRNA and the repeat:anti-repeat duplex of the PAM duplex.

[0110] As used herein, the term "PAM interaction domain" generally refers to a domain that interacts with the protospacer adjacent motif (PAM) outside the seed sequence in the region targeted by Cas protein. Examples of PAM interaction domains include, but are not limited to, the topoisomerase homology (TOPO) domain and C-terminal domain (CTD) present in Cas protein.

[0111] Any variant of the enzymes described herein with 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 a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by replacing 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 are mutated between species without changing the basic function of the encoded protein. Such conservatively substituted variants may include variants having at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the endonuclease protein sequences described herein (e.g., an MG44, MG46, MG71, MG72, MG73, MG74, MG86, MG87, MG88, or MG89 family endonucleases, or any family nuclease described herein). In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can include sequences with substitutions such that the activity of one or more important active site residues or guide RNA binding residues of the endonuclease is not destroyed. In some embodiments, functional variants of any of the proteins described herein lack at least one substitution of a conserved or functional residue in the RuvC or HNH domain of the endonucleases described herein.In some embodiments, a functional variant of any of the proteins described herein lacks all substitutions of conserved or functional residues of the RuvC or HNH domains of the endonucleases described herein.

[0112] Conservative substitution tables providing functionally similar amino acids are available in a variety of references (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)). Each of the following eight groups contains 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).

[0113] overview

[0114] Discovery of new Cas enzymes with unique functionality and structure could further disrupt deoxyribonucleic acid (DNA) editing technology, endowing it with 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 microbes and the net diversity of microbial species, a relatively small number of functionally characterized CRISPR / Cas enzymes exist in the literature. This is in part because the vast number of microbial species are not easily cultured under laboratory conditions. Metagenomic sequencing from natural environmental niches representing a large number of microbial species could dramatically increase the number of known new CRISPR / Cas systems, endowing it with the potential to expedite the discovery of new oligonucleotide editing functions. A fruitful recent example of such an approach is illustrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microbial communities.

[0115] 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 repetitive sequences (30-40 bp) separated by equally short spacer sequences that encode RNA-based targeting elements; and (ii) an ORF encoding a Cas that 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 generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seed) and the crRNA guide; and (ii) the presence of a protospacer adjacent motif (PAM) sequence within a defined vicinity of the target seed (PAM is usually a sequence that is not commonly represented in the host genome). Depending on the exact function and composition of the system, CRISPR-Cas systems are commonly organized into two classes, five types, and 16 subtypes based on shared functional features and evolutionary similarities.

[0116] Class I CRISPR-Cas systems have large, multi-subunit effector complexes and include types I, III, and IV.

[0117] 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 elements to release a short mature crRNA that directs the nuclease complex to the nucleic acid target, followed by a suitable short consensus sequence called the protospacer adjacent motif (PAM). This processing occurs via the endoribonuclease subunit (Cas6) of a large 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.

[0118] Type III CRISPR systems can be characterized by the presence of a central nuclease known as Cas10, along with repeat-associated mysterious proteins (RAMPs) that contain 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).

[0119] Type IV CRISPR-Cas systems possess an effector complex consisting of 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 commonly present on endogenous plasmids.

[0120] Class II CRISPR-Cas systems generally have single-polypeptide multi-domain nuclease effectors and include types II, V and VI.

[0121] 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 special endonuclease subunit, but rather a small transcoding crRNA (tracrRNA) with a region complementary to the array repeat sequence, which interacts with both its corresponding effector nuclease (e.g., Cas9) and the repeat sequence to form a precursor dsRNA structure and is cleaved by endogenous RNAse III to generate a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nucleases are known as DNA nucleases. Type 2 effectors generally exhibit a structure consisting of a RuvC-like endonuclease domain that fits into an RNase H fold with an unrelated HNH nuclease domain inserted into the fold of the RuvC-like nuclease domain. The RuvC-like domain is involved in cleavage of the target (e.g., crRNA-complementary) DNA strand, while the HNH domain is involved in cleavage of the replacement DNA strand. Type II effectors can also contain a PAM-interacting domain or PI domain, which contains a TOPO region and a CTD region that contribute to the recognition of a protospacer adjacent motif (PAM) site in the vicinity of the crRNA-targeted DNA region.

[0122] 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 tracrRNA to process pre-crRNA into mature crRNA, but unlike type II systems that require RNAse III to cleave pre-crRNA into multiple crRNAs, type V systems can cleave pre-crRNA using the effector nuclease itself. Like type II RISPR-Cas systems, type V CRISPR-Cas systems are known DNA nucleases. Unlike type II CRISPR-Cas systems, some type V enzymes (e.g., Cas12a) appear to have robust single-stranded non-specific deoxyribonuclease activity that is activated by the first crRNA-directed cleavage of the double-stranded target sequence.

[0123] Type VI CRIPSR-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 do not appear to require tracrRNA to process pre-crRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) appear to have robust single-stranded non-specific nuclease (ribonuclease) activity that is activated by the first crRNA-directed cleavage of the target RNA.

[0124] Class II CRISPR-Cas are simpler constructs and therefore have been the most widely applied in engineering and development as engineered nuclease / genome editing applications.

[0125] One early application of such a system for in vitro use can be found in Jinek et al. (Science. 2012 Aug 17;337(6096):816-21, incorporated herein by reference in its entirety). The Jinek study first combined (i) recombinantly expressed, purified full-length Cas9 (e.g., class II, type II Cas enzyme) isolated from Streptococcus pyogenes SF370, (ii) purified mature ∼42 nt crRNA (total crRNA transcribed in vitro from a synthetic DNA template bearing a T7 promoter sequence) that yields a ∼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+ Jinek later described an improved engineered system in which (ii) crRNA is attached 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 (compare the top and bottom panels of FIG. 2).

[0126] Mali et al. (Science. 2013 Feb 15;339(6121):823-826.), which is incorporated herein by reference in its entirety, later adapted this system for use in mammalian cells by providing a DNA vector encoding (i) an ORF encoding a codon-optimized Cas9 (e.g., a class II, 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., 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 a tracrRNA sequence) under a suitable polymerase III promoter (e.g., U6 promoter).

[0127] MG enzyme

[0128] In one aspect, the disclosure provides an engineered nuclease system comprising (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may comprise a RuvC domain or a portion thereof (e.g., a RuvC_I, RuvC_II, or RuvC_III domain). The endonuclease may comprise an HNH domain. The endonuclease may comprise a PAM interaction (PI) domain.

[0129] In some cases, the endonuclease may include a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-549 or 602-1276. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1-549 or 602-1276.

[0130] In some embodiments, an endonuclease system according to the present disclosure may include any of the components set forth in Table 1.

[0131] [Table 1]

[0132] In some cases, the endonuclease may include a variant having one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. The NLS may be added to the N-terminus or C-terminus of any one of SEQ ID NOs: 1-549 or 602-1276, or a variant having at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any one of SEQ ID NOs: 1-549 or 602-1276. The NLS may be an SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS may comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identity to any one of SEQ ID NOs: 586-601. The NLS may comprise a sequence substantially identical to any one of SEQ ID NOs: 586-601. The NLS may comprise any of the sequences in Table 2 below, or a combination thereof.

[0133] [Table 2]

[0134] In one aspect, the disclosure provides an engineered nuclease system comprising (a) an endonuclease configured to have selectivity for a protospacer adjacent motif (PAM) sequence comprising any one of SEQ ID NOs: 550-567, wherein the endonuclease is a class 2 type II Cas endonuclease. In some embodiments, the system further comprises (b) an engineered guide nucleic acid structure configured to form a complex with the endonuclease, the guide nucleic acid structure comprising (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) 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 engineered to bind to a PAM sequence that is different from the native PAM sequence of the endonuclease. 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 80% identity with a Cas9 endonuclease. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the HNH domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease is configured to have selectivity for a PAM sequence comprising any one of SEQ ID NOs: 553, 555, or 566, or a variant thereof. In some embodiments, the engineered guide nucleic acid structure comprises at least two ribonucleic acid polynucleotides. In some embodiments, the engineered guide nucleic acid structure comprises one ribonucleic acid polynucleotide comprising a guide ribonucleic acid sequence and a tracr ribonucleic acid sequence. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or an engineered The constructed guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The guide nucleic acid structure, which may be comprised or engineered, comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the targeting nucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. The NLS comprises a sequence comprising any one of SEQ ID NOs: 586-601, or a variant thereof. In some embodiments, the system further comprises a single-stranded or double-stranded DNA repair template comprising, in a 5' to 3' direction, a first homologous arm comprising a sequence of at least 20 nucleotides 5' to a target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homologous arm comprising a sequence of at least 20 nucleotides 3' to the target sequence. In some embodiments, the first or second homologous arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. In some embodiments, the system further comprises a Mg. 2+In some embodiments, the endonuclease and the tracr ribonucleic acid sequence are derived from distinct bacterial species within the same phylum. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity thereto. In some embodiments, the sequence identity is determined by the BLASTP, CLUSTALW, MUSCLE, MAFFT, or CLUSTALW algorithm with Smith-Waterman homology search algorithm parameters. In some embodiments, the sequence identity is determined by the BLASTP homology search algorithm using the BLOSUM62 scoring matrix with parameters of word length (W) of 3, expectation (E) of 10, and gap cost set at 11 presence, 1 extension, and with a conditional composition score matrix adjustment. In some embodiments, the PAM sequence is 3' to the target deoxyribonucleic acid sequence.

[0135] In some aspects, the disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, the nucleic acid encoding a Class 2 Type II Cas endonuclease configured to be selective for a protospacer adjacent motif (PAM) comprising any one of SEQ ID NOs: 550-567. In some embodiments, the endonuclease comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain.In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the organism is a bacterium, an archaea, a eukaryote, a fungus, a plant, a mammal, or a human organism.

[0136] In some aspects, the disclosure provides an engineered nuclease system comprising: (a) an endonuclease having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1-549, 602-1276, or a variant thereof; and (b) an engineered guide nucleic acid structure, wherein the engineered guide RNA is configured to form a complex with the endonuclease, and the engineered guide RNA comprises a targeting nucleic acid sequence configured to hybridize to a target nucleic acid sequence. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and comprises (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.

[0137] In some embodiments, the disclosure provides a method for the preparation of a tracr sequence having (a) (i) at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662; or (ii) a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 568-585 or 1643-16 44 non-degenerate nucleotides of at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity; and (b) a Class 2 Type II Cas endonuclease configured to bind to the engineered guide nucleic acid structure. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 571, 573, or 584 non-degenerate nucleotides. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and includes (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.In some embodiments, the endonuclease comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1-549, 602-1276, or a variant thereof. In some embodiments, the endonuclease comprises a sequence set forth in any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0138] In some aspects, the disclosure provides engineered guide nucleic acid structures comprising: (a) a targeting nucleic acid sequence comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule; and (b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex, one of which comprises a tracr sequence, wherein the two complementary stretches of nucleotides are covalently linked to each other with an intervening nucleotide, wherein the engineered guide ribonucleic acid polynucleotide is selected from the group consisting of SEQ ID NOs: 1-549, 602-603, 604-605, 606-607, 608-609, 610-611, 612-613, 614-615, 616-617, 618-619, 620-621, 622-623, 624-625, 626-627, 628-629, 630-631, 632-633, 634-635, 636-637, 638-639, 640-641, 642-643, 644-645, 646-647, 648-650, 649-651, 652-653, 654-655, 656-657, 658-659, 660-661, 661-670, 662-671, 663-672, 664-673, 665-674, 666-675, 676-677, 678-679, 679-700, 679-701, 1276, or a variant thereof, and capable of forming a complex with an endonuclease having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the endonuclease and targeting the complex to a target sequence of a target DNA molecule. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least 80% identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584.

[0139] In some aspects, the present disclosure provides an engineered vector comprising any of the nucleic acids described herein. In some embodiments, the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV)-derived virion, a lentivirus, or an adenovirus.

[0140] In some aspects, the present disclosure provides a cell comprising any of the vectors described herein or any of the nucleic acids described herein. In some embodiments, the cell is a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human cell.

[0141] In some aspects, the disclosure provides a method of producing an endonuclease comprising culturing any of the cells described herein.

[0142] In some aspects, the disclosure provides a method of binding, cleaving, marking or modifying a double-stranded deoxyribonucleic acid polynucleotide comprising contacting the double-stranded deoxyribonucleic acid polynucleotide with a Class 2 Type II Cas endonuclease in a complex with an endonuclease and an engineered guide nucleic acid structure configured to bind to the double-stranded deoxyribonucleic acid polynucleotide, wherein the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM), and the PAM comprises a sequence set forth in any one of SEQ ID NOs: 550-567. In some embodiments, the endonuclease comprises a PI domain that includes a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises a RuvC domain. In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the RuvC domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the RuvC domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a RuvC domain of any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof. In some embodiments, the HNH domain has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the HNH domains of SEQ ID NOs: 259, 296, or 484, or a variant thereof.In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or an engineered The constructed guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644.In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661. The engineered guide nucleic acid structure comprises a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 571, 573, or 584. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and comprises (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the targeting nucleic acid sequence is complementary to a bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length.

[0143] In some aspects, the disclosure provides a method of editing the AAVS1 locus in a cell, comprising contacting the cell with: (a) an RNA-guided endonuclease; and (b) an engineered guide nucleic acid structure, wherein the engineered guide nucleic acid structure is configured to form a complex with the endonuclease; and wherein the engineered guide nucleic acid structure comprises a spacer sequence configured to hybridize to a region of the AAVS1 locus, wherein the engineered guide nucleic acid structure comprises a targeting sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least 18 contiguous nucleotides of any one of SEQ ID NOs: 1665-1666, or its reverse complement. In some embodiments, the engineered guide nucleic acid structure has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1663 or 1664. In some embodiments, the engineered guide nucleic acid structure is MG71-2-AAVS1-sgRNA-C3 or MG71-2-AAVS1-sgRNA-E2.

[0144] In some aspects, the disclosure provides a method of editing a TRAC locus in a cell, comprising contacting the cell with: (a) an RNA-guided endonuclease; and (b) an engineered guide nucleic acid structure, wherein the engineered guide nucleic acid structure is configured to form a complex with the endonuclease; and wherein the engineered guide nucleic acid structure comprises a spacer sequence configured to hybridize to a region of the TRAC locus, and wherein the engineered guide nucleic acid structure comprises a targeting sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to at least 18 contiguous nucleotides of any one of SEQ ID NOs: 1668 or 1676-1682, or its reverse complement. In some embodiments, the engineered guide nucleic acid structure has at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1667 or 1669-1675. In some embodiments, the engineered guide nucleic acid structure is MG73-1-TRAC-sgRNA-G3, MG89-2-TRAC-sgRNA-F1, MG89-2-TRAC-sgRNA-G5, MG89-2-TRAC-sgRNA-E5, MG89-2-TRAC-sgRNA-F5, MG89-2-TRAC-sgRNA-G1, MG89-2-TRAC-sgRNA-E1, MG89-2-TRAC-sgRNA-B1. In some embodiments, the engineered guide nucleic acid structure is configured to form a complex with an endonuclease and includes (i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.In some embodiments, the targeting nucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1645-1662, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to any one of the non-degenerate nucleotides of SEQ ID NOs: 568-585 or 1643-1644. In some embodiments, the engineered guide nucleic acid structure comprises a tracr sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1648, 1650, or 1661, or the engineered guide nucleic acid structure comprises a sequence having at least 80% identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 571, 573, or 584. In some embodiments, the RNA-guided endonuclease is a class 2, type II Cas endonuclease. In some embodiments, the endonuclease is configured to have a preference for PAM sequences comprising any one of SEQ ID NOs: 550-567.In some embodiments, the endonuclease comprises a PI domain that includes a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1277-1641 or 1683, or a variant thereof. or the endonuclease further comprises a PI domain having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a PI domain of any one of SEQ ID NOs: 1-549 or 602-1276. In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof.In some embodiments, the endonuclease further comprises a PI domain comprising a sequence having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 259, 296, or 484, or a variant thereof. In some embodiments, the endonuclease comprises any one of SEQ ID NOs: 1, 217, 258, 259, 284, 296, 297, 306, 357, 403, 404, 405, 463, 464, 465, 356, or 484, or a variant thereof having at least 55% identity, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0145] The disclosed system can be used for various applications, such as, for example, binding to nucleic acid molecules (e.g., sequence-specific binding), nucleic acid editing (e.g., gene editing), etc. Such systems can be used, for example, to address (e.g., remove or replace) genetically inherited mutations that may cause disease in a subject, to inactivate genes to confirm their function in cells, as diagnostic tools to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or amplified DNA sequences that code for disease-causing mutations), as inactivated enzymes combined with probes to target specific nucleotide sequences (e.g., sequences that code for antibiotic resistance in bacteria), to inactivate viruses by targeting viral genomes or to prevent them from infecting host cells, to add genes or modify metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites, to establish gene drive elements for evolutionary selection, and as biosensors to detect cellular perturbations by exogenous small molecules and nucleotides.

[0146] [Table 3-1]

[0147] [Table 3-2]

[0148] [Table 3-3]

[0149] [Table 3-4]

[0150] [Table 3-5]

[0151]

Table 3-6

[0152]

Table 3-7

[0153]

Table 3-8

[0154]

Table 3-9

[0155]

Table 3-10

[0156]

Table 3-11

[0157]

Table 3-12

[0158]

Table 3-13

[0159]

Table 3-14

[0160]

Table 3-15

[0161]

Table 3-16

[0162]

Table 3-17

[0163]

Table 3-18

[0164]

Table 3-19

[0165]

Table 3-20

[0166]

Table 3-21

[0167]

Table 3-22

[0168]

Table 3-23

[0169]

Table 3-24

[0170]

Table 3-25

[0171]

Table 3-26

[0172]

Table 3-27

[0173]

Table 3-28

[0174]

Table 3-29

[0175]

Table 3-30

[0176]

Table 3-31

[0177]

Table 3-32

[0178]

Table 3-33

[0179]

Table 3-34

[0180]

Table 3-35

[0181]

Table 3-36

[0182]

Table 3-37

[0183]

Table 3-38

[0184]

Table 3-39

[0185]

Table 3-40

[0186]

Table 3-41

[0187]

Table 3-42

[0188]

Table 3-43

[0189]

Table 3-44

[0190]

Table 3-45

[0191]

Table 3-46

[0192]

Table 3-47

[0193]

Table 3-48

[0194]

Table 3-49

[0195]

Table 3-50

[0196]

Table 3-51

[0197]

Table 3-52

[0198]

Table 3-53

[0199]

Table 3-54

[0200]

Table 3-55

[0201]

Table 3-56

[0202]

Table 3-57

[0203]

Table 3-58

[0204]

Table 3-59

[0205]

Table 3-60

[0206]

Table 3-61

[0207]

Table 3-62

[0208]

Table 3-63

[0209]

Table 3-64

[0210]

Table 3-65

[0211]

Table 3-66

[0212]

Table 3-67

[0213]

Table 3-68

[0214]

Table 3-69

[0215]

Table 3-70

[0216]

Table 3-71

[0217]

Table 3-72

[0218]

Table 3-73

[0219]

Table 3-74

[0220]

Table 3-75

[0221]

Table 3-76

[0222]

Table 3-77

[0223]

Table 3-78

[0224]

Table 3-79

[0225]

Table 3-80

[0226]

Table 3-81

[0227]

Table 3-82

[0228]

Table 3-83

[0229]

Table 3-84

[0230]

Table 3-85

[0231]

Table 3-86

[0232]

Table 3-87

[0233]

Table 3-88

[0234]

Table 3-89

[0235]

Table 3-90

[0236]

Table 3-91

[0237]

Table 3-92

[0238]

Table 3-93

[0239]

Table 3-94

[0240]

Table 3-95

[0241]

Table 3-96

[0242]

Table 3-97

[0243]

Table 3-98

[0244]

Table 3-99

[0245]

Table 3-100

[0246]

Table 3-101

[0247]

Table 3-102

[0248]

Table 3-103

[0249]

Table 3-104

[0250]

Table 3-105

[0251]

Table 3-106

[0252]

Table 3-107

[0253]

Table 3-108

[0254]

Table 3-109

[0255]

Table 3-110

[0256]

Table 3-111

[0257]

Table 3-112

[0258]

Table 3-113

[0259]

Table 3-114

[0260]

Table 3-115

[0261]

Table 3-116

[0262]

Table 3-117

[0263]

Table 3-118

[0264]

Table 3-119

[0265]

Table 3-120

[0266]

Table 3-121

[0267]

Table 3-122

[0268]

Table 3-123

[0269]

Table 3-124

[0270]

Table 3-125

[0271]

Table 3-126

[0272]

Table 3-127

[0273]

Table 3-128

[0274]

Table 3-129

[0275]

Table 3-130

[0276]

Table 3-131

[0277]

Table 3-132

[0278]

Table 3-133

[0279]

Table 3-134

[0280]

Table 3-135

[0281]

Table 3-136

[0282]

Table 3-137

[0283]

Table 3-138

[0284]

Table 3-139

[0285]

Table 3-140

[0286]

Table 3-141

[0287]

Table 3-142

[0288]

Table 3-143

[0289]

Table 3-144

[0290]

Table 3-145

[0291]

Table 3-146

[0292]

Table 3-147

[0293]

Table 3-148

[0294]

Table 3-149

[0295]

Table 3-150

[0296]

Table 3-151

[0297]

Table 3-152

[0298]

Table 3-153

[0299]

Table 3-154

[0300]

Table 3-155

[0301]

Table 3-156

[0302]

Table 3-157

[0303]

Table 3-158

[0304]

Table 3-159

[0305]

Table 3-160

[0306]

Table 3-161

[0307]

Table 3-162

[0308]

Table 3-163

[0309]

Table 3-164

[0310]

Table 3-165

[0311]

Table 3-166

[0312]

Table 3-167

[0313]

Table 3-168

[0314]

Table 3-169

[0315]

Table 3-170

[0316]

Table 3-171

[0317]

Table 3-172

[0318]

Table 3-173

[0319]

Table 3-174

[0320]

Table 3-175

[0321]

Table 3-176

[0322]

Table 3-177

[0323]

Table 3-178

[0324]

Table 3-179

[0325]

Table 3-180

[0326]

Table 3-181

[0327]

Table 3-182

[0328]

Table 3-183

[0329]

Table 3-184

[0330]

Table 3-185

[0331]

Table 3-186

[0332]

Table 3-187

[0333]

Table 3-188

[0334]

Table 3-189

[0335]

Table 3-190

[0336]

Table 3-191

[0337]

Table 3-192

[0338]

Table 3-193

[0339]

Table 3-194

[0340]

Table 3-195

[0341]

Table 3-196

[0342]

Table 3-197

[0343]

Table 3-198

[0344]

Table 3-199

[0345]

Table 3-200

[0346]

Table 3-201

[0347]

Table 3-202

[0348]

Table 3-203

[0349]

Table 3-204

[0350]

Table 3-205

[0351]

Table 3-206

[0352]

Table 3-207

[0353]

Table 3-208

[0354]

Table 3-209

[0355]

Table 3-210

[0356]

Table 3-211

[0357]

Table 3-212

[0358]

Table 3-213

[0359]

Table 3-214

[0360]

Table 3-215

[0361]

Table 3-216

[0362]

Table 3-217

[0363]

Table 3-218

[0364]

Table 3-219

[0365]

Table 3-220

[0366]

Table 3-221

[0367]

Table 3-222

[0368]

Table 3-223

[0369]

Table 3-224

[0370]

Table 3-225

[0371]

Table 3-226

[0372]

Table 3-227

[0373]

Table 3-228

[0374]

Table 3-229

[0375]

Table 3-230

[0376]

Table 3-231

[0377]

Table 3-232

[0378]

Table 3-233

[0379]

Table 3-234

[0380]

Table 3-235

[0381]

Table 3-236

[0382]

Table 3-237

[0383]

Table 3-238

[0384]

Table 3-239

[0385]

Table 3-240

[0386]

Table 3-241

[0387]

Table 3-242

[0388]

Table 3-243

[0389]

Table 3-244

[0390]

Table 3-245

[0391]

Table 3-246

[0392]

Table 3-247

[0393]

Table 3-248

[0394]

Table 3-249

[0395]

Table 3-250

[0396]

Table 3-251

[0397]

Table 3-252

[0398]

Table 3-253

[0399]

Table 3-254

[0400]

Table 3-255

[0401]

Table 3-256

[0402]

Table 3-257

[0403]

Table 3-258

[0404]

Table 3-259

[0405]

Table 3-260

[0406]

Table 3-261

[0407]

Table 3-262

[0408]

Table 3-263

[0409]

Table 3-264

[0410]

Table 3-265

[0411]

Table 3-266

[0412]

Table 3-267

[0413]

Table 3-268

[0414]

Table 3-269

[0415]

Table 3-270

[0416]

Table 3-271

[0417]

Table 3-272

[0418]

Table 3-273

[0419]

Table 3-274

[0420]

Table 3-275

[0421]

Table 3-276

[0422]

Table 3-277

[0423]

Table 3-278

[0424]

Table 3-279

[0425]

Table 3-280

[0426]

Table 3-281

[0427]

Table 3-282

[0428]

Table 3-283

[0429]

Table 3-284

[0430]

Table 3-285

[0431]

Table 3-286

[0432]

Table 3-287

[0433]

Table 3-288

[0434]

Table 3-289

[0435]

Table 3-290

[0436]

Table 3-291

[0437]

Table 3-292

[0438]

Table 3-293

[0439]

Table 3-294

[0440]

Table 3-295

[0441]

Table 3-296

[0442]

Table 3-297

[0443]

Table 3-298

[0444]

Table 3-299

[0445]

Table 3-300

[0446]

Table 3-301

[0447]

Table 3-302

[0448]

Table 3-303

[0449]

Table 3-304

[0450]

Table 3-305

[0451]

Table 3-306

[0452]

Table 3-307

[0453]

Table 3-308

[0454]

Table 3-309

[0455]

Table 3-310

[0456]

Table 3-311

[0457]

Table 3-312

[0458]

Table 3-313

[0459]

Table 3-314

[0460]

Table 3-315

[0461]

Table 3-316

[0462]

Table 3-317

[0463]

Table 3-318

[0464]

Table 3-319

[0465]

Table 3-320

[0466]

Table 3-321

[0467]

Table 3-322

[0468]

Table 3-323

[0469]

Table 3-324

[0470]

Table 3-325

[0471]

Table 3-326

[0472]

Table 3-327

[0473]

Table 3-328

[0474]

Table 3-329

[0475]

Table 3-330

[0476]

Table 3-331

[0477]

Table 3-332

[0478]

Table 3-333

[0479]

Table 3-334

[0480]

Table 3-335

[0481]

Table 3-336

[0482]

Table 3-337

[0483]

Table 3-338

[0484]

Table 3-339

[0485]

Table 3-340

[0486]

Table 3-341

[0487]

Table 3-342

[0488]

Table 3-343

[0489]

Table 3-344

[0490]

Table 3-345

[0491]

Table 3-346

[0492]

Table 3-347

[0493]

Table 3-348

[0494]

Table 3-349

[0495]

Table 3-350

[0496]

Table 3-351

[0497]

Table 3-352

[0498]

Table 3-353

[0499]

Table 3-354

[0500]

Table 3-355

[0501]

Table 3-356

[0502]

Table 3-357

[0503]

Table 3-358

[0504]

Table 3-359

[0505]

Table 3-360

[0506]

Table 3-361

[0507]

Table 3-362

[0508]

Table 3-363

[0509]

Table 3-364

[0510]

Table 3-365

[0511]

Table 3-366

[0512]

Table 3-367

[0513]

Table 3-368

[0514]

Table 3-369

[0515]

Table 3-370

[0516]

Table 3-371

[0517]

Table 3-372

[0518]

Table 3-373

[0519]

Table 3-374

[0520]

Table 3-375

[0521]

Table 3-376

[0522]

Table 3-377

[0523]

Table 3-378

[0524]

Table 3-379

[0525]

Table 3-380

[0526]

Table 3-381

[0527]

Table 3-382

[0528]

Table 3-383

[0529]

Table 3-384

[0530]

Table 3-385

[0531]

Table 3-386

[0532]

Table 3-387

[0533]

Table 3-388

[0534]

Table 3-389

[0535]

Table 3-390

[0536] Example 1 -Metagenomics analysis of novel proteins The metagenomics samples were collected from sediments, soils and animals. Deoxyribonucleic acid (DNA) was extracted using Zymobiomics DNA miniprep kit and sequenced on Illumina HiSeq® 2500. Samples were collected with the consent of the owners. Additional raw sequence data from public sources included animal microbiome, sediment, soil, hot springs, hydrothermal vents, ocean, peat, permafrost and sewage sequences. The metagenomics sequence data was searched using hidden Markov models generated based on known Cas protein sequences, including type II Cas effector proteins. Novel effector proteins identified by the search were aligned against known proteins to identify possible active sites. This metagenomics workflow resulted in the details of class II, type II CRISPR endonucleases of the MG44, MG46, MG71, MG72, MG73, MG74, MG87, MG88 and MG89 families described herein.

[0537] Example 2 - Discovery of the MG44, MG46, MG71, MG72, MG73, MG74, MG87, MG88 and MG89 families of CRISPR systems Analysis of the data from the metagenomics analysis of Example 1 revealed a new cluster of putative CRISPR systems, including nine families (MG44, MG46, MG71, MG72, MG73, MG74, MG87, MG88 and MG89), not previously described. The corresponding protein and nucleic acid sequences of these novel enzymes and their exemplary subdomains are presented as SEQ ID NOs: 1-549 or 602-1276.

[0538] Example 3 - Determination of protospacer-adjacent motifs The PAM sequence was determined by sequencing a plasmid containing a randomly generated PAM sequence that could be cleaved by a putative endonuclease expressed in an E. coli lysate-based expression system (myTXTL, Arbor Biosciences). In this system, an E. coli codon-optimized nucleotide sequence was transcribed and translated from a PCR fragment under the control of a T7 promoter. A second PCR fragment with a tracr sequence under a T7 promoter and a minimal CRISPR array consisting of a T7 promoter followed by a repeat spacer repeat sequence were transcribed in the same reaction. Successful expression of the endonuclease sequence and the tracr sequence in the TXTL system, followed by CRISPR array processing, yielded active in vitro CRISPR nuclease complexes.

[0539] A library of target plasmids containing a spacer sequence matching that in the minimal array followed by 8N mixed bases (putative PAM sequence) was incubated with the products of the TXTL reaction. After 1-3 hours, the reaction was stopped and DNA was recovered via a DNA clean-up kit, e.g., Zymo DCC, AMPure XP beads, QiaQuick, etc. The adapter sequence was blunt-end ligated to DNA with an active PAM sequence that had been cleaved by the endonuclease, while uncleaved DNA was inaccessible for ligation. A DNA segment containing the active PAM sequence was then amplified by PCR using primers specific for the library and adapter sequence. The PCR amplification products were resolved on a gel to identify amplicons corresponding to the cleavage events. The amplified segments of the cleavage reaction were also used as templates for the preparation of an NGS library. Sequencing this resulting library, a subset of the starting 8N library, revealed sequences containing the correct PAM for the active CRISPR complex. For PAM testing with single RNA constructs, the same procedure was repeated except that in vitro transcribed RNA was added along with the plasmid library and the tracr / minimal CRISPR array template was omitted. For endonucleases for which NGS libraries were prepared, seqLogo (e.g., Huber et al. Nat Methods. 2015 Feb;12(2):115-21) representations were constructed and shown in Figure 3. The seqLogo module used to construct these representations takes a position weight matrix of DNA sequence motifs (e.g., PAM sequences) and plots the corresponding sequence logos as introduced by Schneider and Stephens (see, e.g., Schneider et al. Nucleic Acids Res. 1990 Oct 25;18(20):6097-100). Letters representing sequences in the seqLogo representation were superimposed on top of each other for each position in the aligned sequences (e.g., PAM sequences). The height of each letter is proportional to its frequency, and the letters are sorted so that the most common letters are at the top.

[0540] Example 4 - Predicted in vitro cleavage efficiency of MG CRISPR complexes The endonuclease is expressed as a His-tagged fusion protein from an inducible T7 promoter in a protease-deficient E. coli B strain. Cells expressing the His-tagged protein are lysed by sonication and the His-tagged protein is purified by Ni-NTA affinity chromatography on a HisTrap FF column (GE Lifescience) on an AKTA Avant FPLC (GE Lifescience). The eluent is separated by SDS-PAGE on an acrylamide gel (Bio-Rad) and stained with InstantBlue Ultrafast Coomassie (Sigma-Aldrich). Purity is determined using densitometry of the protein bands with ImageLab software (Bio-Rad). The purified endonuclease is dialyzed into a storage buffer consisting of 50 mM Tris-HCl, 300 mM NaCl, 1 mM TCEP, 5% glycerol, pH 7.5, and stored at -80°C.

[0541] A target DNA containing a spacer sequence and a PAM sequence (e.g., as determined in Example 3) is constructed by DNA synthesis. A single representative PAM is selected for testing when the PAM has degenerate bases. The target DNA consists of 2200 bp linear DNA derived from a plasmid via PCR amplification with the PAM and a spacer located 700 bp from one end. Successful cleavage results in fragments of 700 and 1500 bp. The target DNA, in vitro transcribed single RNA, and purified recombinant protein are combined in cleavage buffer (10 mM Tris, 100 mM NaCl, 10 mM MgCl2) with excess protein and RNA and incubated for 5 min to 3 h, typically 1 h. The reaction is stopped via the addition of RNAse A and a 60 min incubation. The reaction is then resolved on a 1.2% TAE agarose gel and the fraction of cleaved target DNA is quantified with ImageLab software.

[0542] Example 5 - (Predicted) Testing of Genome Cleavage Activity of MG CRISPR Complex in E. coli E. coli lacks the ability to efficiently repair double-stranded DNA breaks. Thus, genomic DNA breaks can be a lethal event. Taking advantage of this phenomenon, endonuclease activity is tested in E. coli by recombinantly expressing the endonuclease and tracrRNA in a target strain with the spacer / target sequence and PAM sequence integrated into its genomic DNA.

[0543] In this assay, the PAM sequence is specific for the endonuclease being tested, as determined by the method described in Example 3. The sgRNA sequence is determined based on the sequence and predicted structure of the tracrRNA. An 8-12 bp (typically 10 bp) repeat-anti-repeat pair is selected, starting from the 5' end of the repeat. The remaining 3' end of the repeat and the 5' end of the tracrRNA are replaced with a tetraloop. Typically, the tetraloop is GAAA, but other tetraloops can be used, especially if the GAAA sequence is predicted to interfere with folding. In these cases, a TTCG tetraloop was used.

[0544] The engineered strain with the PAM sequence integrated into the genomic DNA is transformed with DNA encoding the endonuclease. The transformants are then transformed with 50 ng of single guide RNA that is chemically competent and either specific to the target sequence ("on-target") or non-specific to the target ("non-target"). After heat shock, the transformants are allowed to recover in SOC at 37°C for 2 hours. Nuclease efficiency is then determined by 5-fold dilutions grown on induction medium. Colonies are quantified from the dilution series in triplicate.

[0545] Example 6 - (Predicted) testing of genome cleavage activity of MG CRISPR complexes in mammalian cells To demonstrate targeting and cleavage activity in mammalian cells, the MG Cas effector protein sequence is tested in two mammalian expression vectors, (a) one with a C-terminal SV40 NLS and a 2A-GFP tag, and (b) one without a GFP tag and with two SV40 NLS sequences, one on the N-terminus and one on the C-terminus. In some cases, the nucleotide sequence encoding the endonuclease is codon-optimized for expression in mammalian cells.

[0546] The corresponding single guide RNA sequence (sgRNA) with the targeting sequence appended is cloned into a second mammalian expression vector. The two plasmids are co-transfected into HEK293T cells. 72 hours after co-transfection of the expression plasmid and the sgRNA targeting plasmid into HEK293T cells, DNA is extracted and used for preparation of NGS libraries. The rate of NHEJ is measured via indels in the sequencing of the target site to indicate the targeting efficiency of the enzyme in mammalian cells. At least 10 different target sites were selected to test the activity of each protein.

[0547] Example 7 - Gene editing results at the DNA level of TRAC and AAVS1 in K562 cells Transfection of MG71-2, MG73-1, and MG89-2 mRNA with matching guide RNAs (500 ng mRNA / 150 pmol guide) from Table 4 below was performed into K562 cells (200,000) using a Lonza 4D electroporator. Cells were harvested and genomic DNA was prepared 3 days after transfection. PCR primers suitable for use in NGS-based DNA sequencing were generated, optimized, and used to amplify individual target sequences for each guide RNA. Amplicons were sequenced on an Illumina MiSeq machine and analyzed using a proprietary Python script to measure gene editing (Figure 4).

[0548] [Table 4-1]

[0549] [Table 4-2]

[0550] The disclosed system can be used for various applications, such as, for example, binding to nucleic acid molecules (e.g., sequence-specific binding), nucleic acid editing (e.g., gene editing), etc. Such systems can be used, for example, to address (e.g., remove or replace) genetically inherited mutations that may cause disease in a subject, to inactivate genes to confirm their function in cells, as diagnostic tools to detect disease-causing genetic elements (e.g., via cleavage of reverse-transcribed viral RNA or amplified DNA sequences that code for disease-causing mutations), as inactivated enzymes combined with probes to target specific nucleotide sequences (e.g., sequences that code for antibiotic resistance in bacteria), to inactivate viruses by targeting viral genomes or to prevent them from infecting host cells, to add genes or modify metabolic pathways to engineer organisms to produce valuable small molecules, macromolecules, or secondary metabolites, to establish gene drive elements for evolutionary selection, and as biosensors to detect cellular perturbations by exogenous small molecules and nucleotides.

[0551] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing description, the description and explanation of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is therefore contemplated that the present invention will encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention and its methods and structures within the scope of these claims and their equivalents encompassed thereby.

Claims

1. 1. An engineered nuclease system comprising: (a) an endonuclease configured to have selectivity for a protospacer adjacent motif (PAM) sequence comprising 5'-nnnnCC-3' (SEQ ID NO:566), the endonuclease being a Class 2 Type II Cas endonuclease and comprising a PAM interaction (PI) domain having at least 80% sequence identity to SEQ ID NO:1633; (b) an engineered guide nucleic acid structure configured to form a complex with the endonuclease, (i) a targeting nucleic acid sequence configured to hybridize to a target nucleic acid sequence; and (ii) an engineered guide nucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease.

2. The engineered nuclease system of claim 1, wherein the endonuclease further comprises a RuvC domain, an HNH domain, or both.

3. The engineered nuclease system of claim 2, wherein the endonuclease comprises the RuvC domain, and the RuvC domain comprises an amino acid sequence having at least 80% sequence identity to the RuvC domain of SEQ ID NO:

484.

4. The engineered nuclease system described in claim 3, wherein the RuvC domain comprises a RuvC domain of sequence number 484.

5. The engineered nuclease system of claim 2, wherein the endonuclease comprises the HNH domain, and the HNH domain comprises an amino acid sequence having at least 80% sequence identity to the HNH domain of SEQ ID NO:

484.

6. The engineered nuclease system described in claim 5, wherein the HNH domain comprises an HNH domain of sequence number 484.

7. The engineered nuclease system described in claim 1, wherein the PI domain comprises a sequence having at least 90% sequence identity to sequence number 1633.

8. The engineered nuclease system described in claim 7, wherein the PI domain comprises the sequence of sequence number 1633.

9. The engineered nuclease system described in claim 1, wherein the endonuclease comprises a sequence having at least 80% sequence identity to SEQ ID NO:

484.

10. The engineered nuclease system described in claim 9, wherein the endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO:

484.

11. The engineered nuclease system described in claim 10, wherein the endonuclease comprises the sequence of SEQ ID NO:

484.

12. The engineered nuclease system described in claim 1, wherein the tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to SEQ ID NO:1661.

13. The engineered nuclease system described in claim 12, wherein the tracr ribonucleic acid sequence comprises the sequence of SEQ ID NO: 1661.

14. The engineered nuclease system of claim 1, wherein the engineered guide nucleic acid structure comprises a sequence having at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO:

584.

15. The engineered nuclease system of claim 14, wherein the engineered guide nucleic acid structure comprises a non-degenerate nucleotide of sequence number 584.

16. The engineered nuclease system of claim 1, wherein the targeting nucleic acid sequence comprises a spacer sequence configured to hybridize to a region of the TRAC locus or a region of the AAVS1 locus.

17. The engineered nuclease system of claim 16, wherein the engineered guide nucleic acid structure comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1669-1675.

18. The engineered nuclease system of claim 17, wherein the engineered guide nucleic acid structure comprises any one of SEQ ID NOs: 1669-1675.

19. The engineered nuclease system of claim 1, wherein the target nucleic acid sequence comprises a sequence having at least 80% sequence identity to at least 18 consecutive nucleotides of any one of SEQ ID NOs: 1676-1682 or its reverse complement.

20. The engineered nuclease system of claim 19, wherein the target nucleic acid sequence comprises a sequence having at least 18 consecutive nucleotides of any one of SEQ ID NOs: 1676-1682 or its reverse complement.

21. The engineered nuclease system of claim 1, wherein the endonuclease comprises the sequence of SEQ ID NO:484 and the engineered guide nucleic acid structure comprises the sequence of SEQ ID NO:

584.

22. One or more nucleic acids encoding the engineered nuclease system described in any one of claims 1 to 21.

23. A pharmaceutical composition comprising the engineered nuclease system described in any one of claims 1 to 21.

24. The pharmaceutical composition of claim 23 for use in modifying a target polynucleotide.

25. A method for modifying a target polynucleotide, comprising contacting a cell with the engineered nuclease system described in any one of claims 1 to 21 to edit a genetic locus within the cell.

26. A method for modifying a target polynucleotide comprising contacting one or more of the nucleic acids of claim 22 with a cell to edit a genetic locus within the cell.