JP2022520428A - Enzyme with RUVC domain - Google Patents

Abstract

The present disclosure provides endonuclease enzymes with distinctive domain characteristics, as well as methods of using such enzymes or variants thereof. [Selection diagram] Fig. 1

Description

Mutual Reference This application is filed in US Provisional Application No. 62 / 805,868 entitled "MG1 ENZYMES WITH RUVC DOMAINS" filed February 14, 2019, and filed July 15, 2019. US Provisional Application No. 62 / 874,414 entitled "MG1 ENZYMES WITH RUVC DOMAINS" and US Provisional Application No. 62 / entitled "MG2 ENZYMES CONTAING RUVC DOMAINS" filed on February 14, 2019. Claim the interests of US Provisional Application Nos. 805, 878, and US Provisional Application No. 62 / 805,899 entitled "MG3 ENZYMES WITH RUVC DOMAINS" filed February 14, 2019, each of which is by reference. Fully incorporated herein.

Cas enzymes spread to the prokaryotic immune system (~ 45% bacteria, ~ 84% paleobacilli), along with their associated clustered and regularly arranged short circular sequence repeat (CRISPR) -guided ribonucleic acid (RNA). ) It is a component and appears to play a role in protecting such microorganisms from non-self-nucleic acids such as infectious viruses and plasmids by CRISPR-RNA guided nucleic acid cleavage. Deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, but their CRISPR-related (Cas) proteins are very diverse and vary from nucleic acid to each other. Contains an action domain. Although the CRISPR DNA element was observed as early as 1987, the programmable endonuclease cleavage capacity of the CRISPR / Cas complex has been recognized relatively recently and has been used in a variety of DNA manipulation and gene editing applications. It has led to the use of recombinant CRISPR / Cas systems.

Sequence Listing This application contains a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the ASCII was made on February 13, 2020, and 55921-703_601_SL. The file name is txt and the size is 23,363,113 bytes.

In some embodiments, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system is: (a) an endonuclease comprising a RuvC_III domain and an HNH domain, wherein the endonuclease is. Derived from a refractory nucleic acid, where the endonuclease is a class 2 type II Cas endonuclease, and is configured to form a complex with the endonuclease; (b) the endonuclease. The engineered guide ribonucleic acid structure to which the engineered guide ribonucleic acid structure is: (i) with a guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence; (ii) to the endonuclease. Includes an engineered guide ribonucleic acid structure, including a tracr ribonucleic acid sequence configured to bind. In some embodiments, the RuvC_III domain comprises a sequence having at least 70%, at least 75%, at least 80%, or at least 90% sequence identity to any one of SEQ ID NOs: 1827-3637. ..

In some embodiments, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system has at least 75% sequence identity to any one of: (a) SEQ ID NO: 1827-3637. With an endonuclease containing a RuvC_III domain; (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease: (i). Includes a guide ribonucleic acid structure comprising a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.

In some embodiments, the present disclosure provides an engineered nuclease system such that the engineered nuclease system binds to a protospacer flanking motif (PAM) sequence comprising: (a) SEQ ID NO: 5512-5537. Constituted end nucleases, wherein the end nuclease is a class 2 type II Cas end nuclease with an end nuclease; (b) an operation configured to form a complex with the end nuclease. The guided guide ribonucleic acid structure, wherein the engineered guide ribonucleic acid structure is: (i) a guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence; (ii) to bind to the end nuclease. Includes an engineered guide ribonucleic acid structure, including a constructed tracr ribonucleic acid sequence.

In some embodiments, endonucleases are derived from refractory microorganisms. In some embodiments, the endonuclease has not been engineered to bind to a different PAM sequence. In some embodiments, the endonucleases are Cas9 endonucleases, Cas14 endonucleases, Cas12a endonucleases, Cas12b endonucleases, Cas12c endonucleases, Cas12d endonucleases, Cas12e endonucleases, Cas13a endonucleases, Cas13b endonucleases, Cas13c endonucleases. , Or Cas13d endonuclease. In some embodiments, the endonuclease has less than 80% identity to the Cas9 endonuclease. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the tracr ribonucleic acid sequence is at least 80% sequence identical to about 60-90 contiguous nucleotides selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. Includes sex sequences.

In some embodiments, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system is (a) an engineered guide ribonucleic acid structure and the engineered guide ribonucleic acid structure is: (i). ) A guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; (ii) a tracr ribonucleic acid sequence configured to bind to an endonuclease, wherein the tracr ribonucleic acid sequence is here. With a tracr ribonucleic acid sequence comprising a sequence having at least 80% sequence identity to about 60-90 contiguous nucleotides selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538; Includes an engineered guide ribonucleic acid structure comprising (b) a class 2 type II Cas endonuclease configured to bind to the engineered guide ribonucleic acid. In some embodiments, the endonuclease is configured to bind to a protospacer flanking motif (PAM) sequence selected from the group comprising SEQ ID NO: 5512-5537.

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 one ribonucleic acid polynucleotide comprising a guide ribonucleic acid sequence and a tracr ribonucleic acid sequence.

In some embodiments, the guideribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the guideribonucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the endonuclease comprises one or more nuclear localization sequences (NLS) located proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises a sequence selected from SEQ ID NO: 5597-5612.

In some embodiments, the engineered nuclease system comprises a first homology arm comprising a sequence of at least 20 nucleotides in 5'of the target deoxyribonucleic acid sequence from 5'to 3'and at least 10 nucleotides. It further comprises a single-stranded or double-stranded DNA repair template comprising a synthetic DNA sequence and a second homology arm comprising a sequence of at least 20 nucleotides in 3'of the target deoxyribonucleic acid sequence. In some embodiments, the first or second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.

In some embodiments, the engineered nuclease system further comprises a source of Mg ²⁺ .

In some embodiments, the endonuclease and tracr ribonucleic acid sequences are derived from distinct bacterial species within the same phylum. In some embodiments, the endonuclease is derived from a bacterium belonging to the genus Dermabacter. In some embodiments, the endonuclease is derived from a bacterium belonging to the phylum Verrucomicrobiota, the phylum Candidatus Peregrinibacteria, or the phylum Candidatus Melainabacteria. In some embodiments, the endonuclease is derived from a bacterium containing a 16S rRNA gene having at least 90% identity to any one of SEQ ID NOs: 5592-5595.

In some embodiments, the HNH domain comprises a sequence having at least 70% or at least 80% identity to any one of SEQ ID NOs: 5638-5460. In some embodiments, the endonuclease comprises SEQ ID NO: 1-1826 or a variant thereof having at least 55% identity to them. In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1827-1830 or SEQ ID NO: 1827-2140. include.

In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3638-3461 or SEQ ID NO: 3638-3954. include. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5615-5632. In some embodiments, the endonuclease is a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1-4 or SEQ ID NO: 1-319. include.

In some embodiments, the guide RNA structure is at least 70%, 80% relative to a sequence selected from the group consisting of SEQ ID NO: 5461-5464, SEQ ID NO: 5476-5479, or SEQ ID NO: 5476-5489. , Or sequences that are 90% identical. In some embodiments, the guide RNA structure comprises an RNA sequence that is expected to include a hairpin consisting of a stem and a loop, wherein the stem is at least 10, at least 12, or at least 14 base pair ribos. Includes nucleotides and asymmetric bulges within the four base pairs of the loop.

In some embodiments, the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5512-5515 or SEQ ID NO: 5527-5530.

In some embodiments, (a) the endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1827; (b) the guide RNA structure is SEQ ID NO: : 5461 or contains a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5476; and (c) the endonuclease is SEQ ID NO: 5512 or SEQ ID NO: 5527. Is configured to bind to a PAM containing. In some embodiments, (a) the endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1828; (b) the guide RNA structure is SEQ ID NO: : 5462 or contains a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5477; and (c) the endonuclease is SEQ ID NO: 5513 or SEQ ID NO: 5528. Is configured to bind to a PAM containing. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1829; (b) the guide RNA structure is SEQ ID NO: : 546 or contains a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5478; and (c) the endonuclease is SEQ ID NO: 5514 or SEQ ID NO: :. It is configured to bind to a PAM containing 5259. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1830; (b) the guide RNA structure is SEQ ID NO: : 5464 or contains a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5479; and (c) the endonuclease is SEQ ID NO: 5515 or SEQ ID NO: :. It is configured to bind to a PAM containing 5530.

In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2141-2142 or SEQ ID NO: 2141-2241. include. In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3955-3965 or SEQ ID NO: 3955-4055. include. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5632-5638. In some embodiments, the endonuclease is a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 320-321 or SEQ ID NO: 320-420. include. In some embodiments, the guide RNA structure is at least 70%, 80%, or at least 70%, or 80% of a sequence selected from the group consisting of SEQ ID NO: 5465, SEQ ID NO: 5490-5491, or SEQ ID NO: 5490-5494. Contains sequences that are 90% identical. In some embodiments, the guide RNA structure comprises a tracr ribonucleic acid sequence comprising a hairpin containing at least 8, at least 10, or at least 12 base pair ribonucleotides. In some embodiments, the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5516 and SEQ ID NO: 5531. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2141; (b) the guide RNA structure is SEQ ID NO: 5490. Containing sequences that are at least 70%, 80%, or 90% identical relative to; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5531. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2142; (b) the guide RNA structure is SEQ ID NO: 5465. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5491; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5516.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2245-2246. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4059-4060. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5639-5648. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 424-425. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5498-5499 and SEQ ID NO: 5539. .. In some embodiments, the guide RNA structure is expected to include a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. Includes a sequence, wherein the tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin at 5'to 3'where the first hairpin is more than the second hairpin. Has a long stem.

In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2242-2244 or SEQ ID NO: 2247-2249. include. In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to the sequence selected from the group consisting of SEQ ID NO: 4056-4058 and SEQ ID NO: 4061-4063. include. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5639-5648. In some embodiments, the endonuclease is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 421-423 or SEQ ID NO: 426-428. include. In some embodiments, the guide RNA structure is at least for a sequence selected from the group consisting of SEQ ID NO: 5466-5467, SEQ ID NO: 5495-5497, SEQ ID NO: 5500-5502, and SEQ ID NO: 5539. Contains sequences that are 70%, 80%, or 90% identical. In some embodiments, the guide RNA structure is expected to include a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. Includes a sequence, wherein the tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin at 5'to 3'where the first hairpin is more than the second hairpin. Has a long stem. In some embodiments, the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5517-5518 or SEQ ID NO: 5532-5534. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2247; (b) the guide RNA structure is SEQ ID NO: 5500. Containing sequences that are at least 70%, 80%, or 90% identical relative to; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5517 or SEQ ID NO: 5532. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2248; (b) the guide RNA structure is SEQ ID NO: 5501. Containing sequences that are at least 70%, 80%, or 90% identical relative to; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5518 or SEQ ID NO: 5533. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2249; (b) the guide RNA structure is SEQ ID NO: 5502. Containing sequences that are at least 70%, 80%, or 90% identical relative to; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5534.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2253 or SEQ ID NO: 2253-2481. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4067 or SEQ ID NO: 4067-4295. In some embodiments, the endonuclease comprises a peptide motif of SEQ ID NO: 5649. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 432 or SEQ ID NO: 432-660. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5468 or SEQ ID NO: 5503. In some embodiments, the endonuclease is configured to bind to a PAM containing a sequence selected from the group consisting of SEQ ID NO: 5519. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2253; (b) the guide RNA structure is SEQ ID NO: 5468. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5503; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5519.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to the sequence selected from the group consisting of SEQ ID NO: 2482-2489. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to the sequence selected from the group consisting of SEQ ID NO: 4296-4303. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 661-668. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2490-2498. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to the sequence selected from the group consisting of SEQ ID NO: 4304-4212. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 669-677. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to the sequence selected from the group consisting of SEQ ID NO: 5504.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2499 or SEQ ID NO: 2499-2750. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4313 or SEQ ID NO: 4313-4564. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5650-5667. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 678 or SEQ ID NO: 678-929. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2499; (b) the guide RNA structure is SEQ ID NO: 5469. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5505; and (c) the endonuclease binds to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535. It is composed.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2751 or SEQ ID NO: 2751-2913. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4565 or SEQ ID NO: 4565-4727. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5668-5678. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 930 or SEQ ID NO: 930-1092. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5470 or SEQ ID NO: 5506. In some embodiments, the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5521 or SEQ ID NO: 5536. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2751; (b) the guide RNA structure is SEQ ID NO: 5470. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5506; and (c) the endonuclease binds to a PAM comprising SEQ ID NO: 5521 or SEQ ID NO: 5536. It is composed.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2914 or SEQ ID NO: 2914-3174. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4728 or SEQ ID NO: 4728-4988. In some embodiments, the endonuclease comprises at least one, at least two, or at least three peptide motifs selected from the group consisting of SEQ ID NO: 5676-5678. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1093 or SEQ ID NO: 1093-1353. In some embodiments, the guide RNA structure is at least 70%, 80%, or 90% of the sequence selected from the group consisting of SEQ ID NO: 5471, SEQ ID NO: 5507, and SEQ ID NO: 5540-5542. Contains sequences that are identical. In some embodiments, the guide RNA structure comprises a tracr ribonucleic acid sequence that is expected to contain at least two hairpins containing less than five base pair ribonucleotides. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5522. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2914; (b) the guide RNA structure is SEQ ID NO: 5471. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5507; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5522.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3175 or SEQ ID NO: 3175-3330. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4989 or SEQ ID NO: 4989-5146. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5679-5686. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1354 or SEQ ID NO: 1354-1511. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5472 or SEQ ID NO: 5508. In some embodiments, the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5523 or SEQ ID NO: 5537. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3175; (b) the guide RNA structure is SEQ ID NO: 5472. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5508; and (c) the endonuclease binds to a PAM comprising SEQ ID NO: 5523 or SEQ ID NO: 5537. It is composed.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3331 or SEQ ID NO: 3331-3474. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5147 or SEQ ID NO: 5147-5290. In some embodiments, the endonuclease is at least one, at least two, at least three, at least four, or at least five selected from the group consisting of SEQ ID NO: 5674-5675 and SEQ ID NO: 5687-5693. Contains two peptide motifs. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1512 or SEQ ID NO: 1512-1655. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5473 or SEQ ID NO: 5509. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5524. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3331; (b) the guide RNA structure is SEQ ID NO: 5473. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5509; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5524.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3475 or SEQ ID NO: 3475-3568. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5291 or SEQ ID NO: 5291-5389. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5694-569. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1656 or SEQ ID NO: 1656-1755. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5474 or SEQ ID NO: 5510. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5525. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3475; (b) the guide RNA structure is SEQ ID NO: 5474. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5510; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5525.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3569 or SEQ ID NO: 3569-3637. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5390 or SEQ ID NO: 5390-5460. In some embodiments, the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5700-5717. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1756 or SEQ ID NO: 1756-1826. In some embodiments, the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5475 or SEQ ID NO: 5511. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5526. In some embodiments, (a) the endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3569; (b) the guide RNA structure is SEQ ID NO: 5475. Alternatively, it comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5511; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5526. In some embodiments, sequence identity is determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, or Smith-Waterman homology search algorithms. In some embodiments, sequence identity uses parameters from the BLASTUM62 scoring matrix that set the gap cost at 3 wordlength (W), 10 extraction (E) parameters, and 11 extensions and 1 extensions. And, as well as using a conditional compositional score matrix adjustment, it is determined by the BLASTP homology search algorithm.

In some embodiments, the present disclosure provides an engineered guide ribonucleic acid polynucleotide, wherein the engineered guide ribonucleic acid polynucleotide comprises: (a) a DNA target comprising a nucleotide sequence complementary to the target sequence in the target DNA molecule. A protein binding segment comprising two complementary stretches of a nucleotide that hybridizes with a modified segment to form a double-stranded RNA (dsRNA) duplex, wherein the two complements of the above nucleotide. Stretches covalently bind to each other at intervening nucleotides, wherein the engineered guide ribonucleic acid polynucleotide has at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637. It is configured to form a complex with an endonuclease containing the above complex and target the complex to the target sequence of the target DNA molecule. In some embodiments, the DNA targeting segment is located at 5'both of the two complementary stretches of the nucleotide.

In some embodiments, (a) the protein binding segment is at least 70%, at least 80%, or at least 90 of the sequence selected from the group consisting of SEQ ID NO: 5476-5479 or SEQ ID NO: 5476-5489. Includes a sequence having% identity; (b) the protein binding segment is at least relative to a sequence selected from the group consisting of (SEQ ID NO: 5490-5491 or SEQ ID NO: 5490-5494) and SEQ ID NO: 5538. Containing sequences with 70%, at least 80%, or at least 90% identity; (c) protein binding segments are at least 70%, at least 70%, at least relative to the sequence selected from the group consisting of SEQ ID NO: 5498-5499. Containing sequences with 80%, or at least 90% identity; (d) the protein binding segment is at least 70 relative to the sequence selected from the group consisting of SEQ ID NO: 5495-5497 and SEQ ID NO: 5500-5502. Containing sequences having%, at least 80%, or at least 90% identity; (e) the protein binding segment has at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5503. Containing the sequence having; (f) the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5504; (g) the protein binding segment comprises the sequence. Includes sequences having at least 70%, at least 80%, or at least 90% identity to number: 5505; (h) protein binding segments are at least 70%, at least 80%, to SEQ ID NO: 5506. Or contains a sequence having at least 90% identity; (i) the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5507; ( j) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5508; (k) the protein binding segment to SEQ ID NO: 5509. Containing sequences with at least 70%, at least 80%, or at least 90% identity; (l) protein binding segments are at least 70% less than SEQ ID NO: 5510. Both contain sequences having 80%, or at least 90% identity; or (m) the protein binding segment has at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5511. Includes sequences with.

In some embodiments, (a) a guide ribonucleic acid polynucleotide comprises an RNA sequence comprising a hairpin comprising a stem and a loop, wherein the stem is at least 10, at least 12, or at least 14 base pairs. Includes ribonucleotides and asymmetric bulges within the four base pairs of the loop; (b) Guided ribonucleic acid polynucleotides are expected to include hairpins containing at least 8, at least 10, or at least 12 base pairs of ribonucleotides. Containing the tracr ribonucleic acid sequence; (c) the guide ribonucleic acid polynucleotide comprises a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. It comprises the expected guide ribonucleic acid sequence, wherein the tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin in 5'to 3', where the first hairpin is the first. It has a stem longer than 2 hairpins; or (d) the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence that is expected to contain at least 2 hairpins containing ribonucleotides of less than 5 base pairs.

In some embodiments, the present disclosure provides a deoxyribonucleic acid polynucleotide that encodes one of the engineered guide ribonucleic acid polynucleotides described herein.

In some embodiments, the present disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid is a class 2 comprising a RuvC_III domain and an HNH domain. Encodes the type II Cas endonuclease of, where the class 2 type II Cas endonuclease is derived from a refractory microorganism.

In some embodiments, the present disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid is any one of SEQ ID NOs: 1827-3637. It encodes an endonuclease containing a RuvC_III domain with at least 70% sequence identity to one. In some embodiments, the endonuclease comprises an HNH domain having at least 70% or at least 80% sequence identity to any one of SEQ ID NOs: 3638-5460. In some embodiments, the endonuclease comprises SEQ ID NO: 5571-5591 or a variant thereof having at least 70% sequence identity relative to them. In some embodiments, the endonuclease comprises a sequence encoding one or more nuclear localization sequences (NLS) located proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS comprises a sequence selected from SEQ ID NO: 5597-5612.

In some embodiments, the organism is a prokaryote, bacterium, eukaryote, fungus, plant, mammal, rodent, or human. In some embodiments, the organism is E. coli, and: (a) the nucleic acid sequence is at least 70%, 80%, or 90% of the sequence selected from the group consisting of SEQ ID NO: 5571-5575. Have identity; (b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to the sequence selected from the group consisting of SEQ ID NO: 5576-5571; (c) nucleic acid sequence. Has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5578-5580; (d) Nucleic acid sequence is at least relative to SEQ ID NO: 5581. Has 70%, 80%, or 90% identity; (e) Nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5582; (f) Nucleic acid sequence. Has at least 70%, 80%, or 90% identity to SEQ ID NO: 5583; (g) the nucleic acid sequence is at least 70%, 80%, or 90% to SEQ ID NO: 5584. Have identity; (h) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5585; (i) the nucleic acid sequence has at least 70% to SEQ ID NO: 5586. It has 70%, 80%, or 90% identity; or (j) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5587. In some embodiments, the organism is human and: (a) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5588 or SEQ ID NO: 5589; Alternatively, (b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5590 or SEQ ID NO: 5591.

In some embodiments, the present disclosure provides a vector comprising a nucleic acid sequence encoding a class 2 type II Cas endonuclease comprising a RuvC_III domain and an HNH domain, wherein the endonuclease is here for refractory microorganisms. Derived from.

In some embodiments, the present disclosure provides a vector comprising any of the nucleic acids described herein. In some embodiments, the vector further comprises a nucleic acid encoding an engineered guide ribonucleic acid structure configured to form a complex with an endonuclease, wherein the engineered guide ribonucleic acid structure is: (a) target. It comprises a guide ribonucleic acid sequence configured to hybridize to a deoxyribonucleic acid sequence; (b) a tracr ribonucleic acid sequence configured to bind to an endonuclease. In some embodiments, the vector is a plasmid, minicircle, CELiD, adeno-associated virus (AAV) -derived virion, or lentivirus.

In some embodiments, the present disclosure provides cells comprising any of the vectors described herein.

In some embodiments, the disclosure provides a method of producing an endonuclease, which method comprises culturing any of the cells described herein.

In some embodiments, the present disclosure provides methods for binding, cleaving, labeling, or modifying double-stranded deoxyribonucleic acid polynucleotides, wherein: (a) Class 2 type II Cas endonuclease and The step of contacting the double-stranded deoxyribonucleic acid polynucleotide with a class 2 type II Cas endonuclease that forms a complex with an engineered guide ribonucleic acid structure configured to bind to the double-stranded deoxyribonucleic acid polynucleotide. (B) Here, the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer flanking motif (PAM); (c) the PAM is from SEQ ID NO: 5512-5526 or SEQ ID NO: 5527-5537. Contains sequences selected from the group of In some embodiments, the double-stranded deoxyribonucleic acid polynucleotide comprises a first strand comprising a sequence complementary to the sequence of the engineered guide ribonucleic acid structure and a second strand comprising PAM. In some embodiments, the PAM is directly flanking the 3'end of a sequence complementary to the sequence of the engineered guide ribonucleic acid structure.

In some embodiments, class 2 type II Cas endonucleases are Cas9 endonucleases, Cas14 endonucleases, Cas12a endonucleases, Cas12b endonucleases, Cas12c endonucleases, Cas12d endonucleases, Cas12e endonucleases, Cas13a endonucleases, Cas13b. Not an endonuclease, Cas13c endonuclease, or Cas13d endonuclease. In some embodiments, class 2 type II Cas endonucleases are derived from refractory microorganisms. In some embodiments, the double-stranded deoxyribonucleic acid polynucleotide is a double-stranded deoxyribonucleic acid polynucleotide of eukaryote, plant, fungus, mammal, rodent, or human.

In some embodiments, (a) PAM comprises a sequence selected from the group consisting of SEQ ID NO: 5512-5515 and SEQ ID NO: 5527-5530; (b) PAM comprises SEQ ID NO: 5516 or SEQ ID NO: :. Includes 5531; (c) PAM comprises SEQ ID NO: 5539; (d) PAM comprises SEQ ID NO: 5517 or SEQ ID NO: 5518; (e) PAM comprises SEQ ID NO: 5519; (f) PAM comprises. SEQ ID NO: 5520 or SEQ ID NO: 5535; (g) PAM comprises SEQ ID NO: 5521 or SEQ ID NO: 5536; (h) PAM comprises SEQ ID NO: 5522; (i) PAM comprises SEQ ID NO: 5523. Alternatively, the SEQ ID NO: 5537 is included; (j) PAM comprises SEQ ID NO: 5524; (k) PAM comprises SEQ ID NO: 5525; or (l) PAM comprises SEQ ID NO: 5526.

In some embodiments, the present disclosure provides a method for modifying a target nucleic acid locus, wherein the method incorporates any of the engineered nuclease systems described herein into the target nucleic acid locus. Containing a step of delivery, where the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure, where the complex is associated with the binding of the complex to the target nucleic acid locus. , The complex is configured to modify the target nucleic acid locus. In some embodiments, modifying the target nucleic acid locus comprises binding, nicking, cleaving, and labeling the target nucleic acid locus. In some embodiments, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. In some embodiments, the target nucleic acid locus is in vitro. In some embodiments, the target nucleic acid locus is intracellular. In some embodiments, the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell.

In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus delivers the nucleic acid of any of claims 135-140 or the vector of any of claims 142-146. Including doing. In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a nucleic acid comprising an open reading frame encoding an endonuclease. In some embodiments, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises delivering a capped mRNA containing an open reading frame encoding an endonuclease. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises delivering the translated polypeptide. In some embodiments, the engineered nuclease system to the target nucleic acid locus is a deoxyribonucleic acid (DNA) that encodes an engineered guide ribonucleic acid structure that is operably linked to the ribonucleic acid (RNA) pol III promoter. Includes delivery. In some embodiments, endonucleases cause single-strand or double-strand breaks at or proximal to the target locus.

Further aspects and advantages of the present disclosure will be readily apparent to those of skill in the art from the detailed description below, wherein only exemplary embodiments of the present disclosure are shown and described. As will be appreciated, this disclosure is possible in other embodiments as well as in different embodiments, the various details of which may be modified in various obvious ways without departing from the present disclosure. can. Therefore, the drawings and descriptions are by nature considered exemplary and not limiting.

Incorporation by Reference All publications, patents, and patent applications referred to herein are as if the individual publications, patents, or patent applications are specifically and individually indicated to be incorporated by reference. , To the same extent incorporated herein by reference.

The novel features of the invention are specified, among other things, within the appended claims. A better understanding of the features and advantages of the invention is the following detailed description illustrating exemplary embodiments in which the principles of the invention are used and the following accompanying drawings (in the present specification, "Figure" and ". It will be obtained by referring to FIG. ")").

Shows typical tissues of CRISPR / Cas loci of various classes and types. Shown is the structure of a native Class 2 / II type crRNA / tracrRNA pair compared to a hybrid sgRNA to which both are bound. A conceptual diagram showing the composition of the CRISPR locus encoding an enzyme from the MG1 family is shown. A conceptual diagram showing the composition of the CRISPR locus encoding an enzyme from the MG2 family is shown. A conceptual diagram showing the composition of the CRISPR locus encoding an enzyme from the MG3 family is shown. An alignment based on the structure of the enzyme of the present disclosure (MG1-1) with respect to Cas9 (SEQ ID NO: 5613) from Staphylococcus aureus is shown. An alignment based on the structure of the enzyme of the present disclosure (MG2-1) with respect to Cas9 (SEQ ID NO: 5613) from Staphylococcus aureus is shown. The alignment based on the structure of the enzyme of the present disclosure (MG3-1) with respect to Cas9 (SEQ ID NO: 5614) from Actinomyces naeslundii is shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. Alignments based on the structure of the MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3) are shown. In vitro cleavage of DNA by MG1-4 complexing with its corresponding sgRNA containing targeting sequences of various lengths is shown. A cell cleavage of E. coli genomic DNA using MG1-4 and its corresponding sgRNA is shown. Diluted sequences of MG1-4 transformed cells with targeted or non-targeted spacers are shown (top); lower panel shows quantified data, left bar represents non-targeted sgRNA, right bar Represents the target sgRNA. HEK cells using the MG1-4 or MG1-6 constructs described in Example 11 with their corresponding sgRNAs containing various different targeting sequences targeting different locations in the human genome. It shows the cell indel formation produced by transfection. Vitro cleavage of DNA by MG3-6 forming a complex with its corresponding sgRNA containing targeting sequences of various lengths is shown. A cell cleavage of E. coli genomic DNA using MG3-7 and its corresponding sgRNA is shown. Diluted sequences of MG3-7 transformed cells with targeted or non-targeted spacers are shown (top); bottom panel shows quantified data, left bar represents non-targeted sgRNA, right bar Represents the target sgRNA. Generated by transfection of HEK cells with the MG3-7 construct described in Example 13 together with their corresponding sgRNAs containing various different targeting sequences targeting different positions in the human genome. Shows cell indel formation. In vitro cleavage of DNA by MG15-1, which forms a complex with its corresponding sgRNA containing targeting sequences of various lengths, is shown. Shown is an agarose gel showing the results of cleavage of the PAM vector library in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Shown is an agarose gel showing the results of cleavage of the PAM vector library in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Shown is an agarose gel showing the results of cleavage of the PAM vector library in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Shown is an agarose gel showing the results of cleavage of the PAM vector library in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The expected structure of the corresponding sgRNA of the MG enzyme described herein (eg, as in Example 7) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. The seqLogo representation of the PAM sequence derived via NGS described herein (eg, described in Example 6) is shown. A cell cleavage of E. coli genomic DNA using MG2-7 and its corresponding sgRNA is shown. Diluted sequences of MG2-7 transformed cells with targeted or non-targeted spacers are shown (top); bottom panel shows quantified data, right bar represents non-targeted sgRNA, left bar Represents the target sgRNA. A cell cleavage of E. coli genomic DNA using MG14-1 and its corresponding sgRNA is shown. The dilution series of cells transformed with MG14-1 with the target spacer or non-target spacer is shown (top); the lower panel shows the quantified data, the right bar represents the non-target sgRNA, and the left bar shows. Represents the target sgRNA. A cell cleavage of E. coli genomic DNA using MG15-1 and its corresponding sgRNA is shown. Diluted sequences of MG15-1 transformed cells with targeted or non-targeted spacers are shown (top); bottom panel shows quantified data, right bar represents non-targeted sgRNA, left bar Represents the target sgRNA.

Brief Description of Sequence Listing The sequence listing, filed with this specification, provides exemplary polynucleotide and polypeptide sequences used in the methods, compositions, and systems of the present disclosure. The following is an exemplary description of the sequences in the sequence listing.

MG1

SEQ ID NO: 1-319 show the full length peptide sequence of MG1 nuclease.

SEQ ID NO: 1827-2140 shows the peptide sequence of the RuvC_III domain of the above MG1 nuclease.

SEQ ID NO: 3638-3955 represent peptides in the HNH domain of the above MG1 nuclease.

SEQ ID NO: 5476-5479 represents the nucleotide sequence of MG1 tracrRNA derived from the same locus as MG1 nuclease above (eg, the same locus as SEQ ID NO: 1-4, respectively).

SEQ ID NO: 5461-5464 represent the nucleotide sequence of the sgRNA engineered to function with the MG1 nuclease (eg, SEQ ID NO: 1-4, respectively), where Ns represents the nucleotide of the targeting sequence.

SEQ ID NO: 5571-5575 represents the nucleotide sequence of the E. coli codon-optimized coding sequence of the MG1 family enzyme (SEQ ID NO: 1-4).

SEQ ID NO: 5588-5589 represent the nucleotide sequence of the human codon-optimized coding sequence for the MG1 family of enzymes (SEQ ID NOs: 1 and 3).

SEQ ID NO: 5616-5632 show the peptide motif properties of the MG1 family of enzymes.

MG2

SEQ ID NO: 320-420 shows the full length peptide sequence of MG2 nuclease.

SEQ ID NO: 2141-2241 shows the peptide sequence of the RuvC_III domain of the above MG2 nuclease.

SEQ ID NO: 3955-4055 represent peptides in the HNH domain of the above MG2 nuclease.

SEQ ID NO: 5490-5494 represents the nucleotide sequence of MG2 tracrRNA derived from the same locus as MG2 nuclease above (eg, the same locus as SEQ ID NOs: 320, 321, 323, 325, and 326, respectively).

SEQ ID NO: 5465 represents the nucleotide sequence of the sgRNA engineered to function with the MG2 nuclease (eg, SEQ ID NO: 321 above).

SEQ ID NO: 5571-5575 represents the nucleotide sequence of the E. coli codon-optimized coding sequence of the MG2 family of enzymes.

SEQ ID NO: 5631-5638 show peptide sequence characteristics of MG2 family enzymes.

MG3

SEQ ID NO: 421-431 shows the full length peptide sequence of MG3 nuclease.

SEQ ID NO: 2242-2251 shows the peptide sequence of the RuvC_III domain of the above MG3 nuclease.

SEQ ID NO: 4056-4066 represent peptides in the HNH domain of the MG3 nuclease above.

SEQ ID NO: 5495-5502 shows the nucleotide sequence of MG3 tracrRNA derived from the same locus as the above MG3 nuclease (eg, the same locus as SEQ ID NO: 421-428, respectively).

SEQ ID NO: 5466-5467 represents the nucleotide sequence of the sgRNA engineered to function with the MG3 nuclease (eg, SEQ ID NO: 421-423).

SEQ ID NO: 5578-5580 show the nucleotide sequence of the E. coli codon-optimized coding sequence of the MG3 family of enzymes.

SEQ ID NO: 5639-5648 show peptide sequence characteristics of MG3 family enzymes.

MG4

SEQ ID NO: 432-660 shows the full-length peptide sequence of MG4 nuclease.

SEQ ID NO: 2253-2481 shows the peptide sequence of the RuvC_III domain of the above MG4 nuclease.

SEQ ID NO: 4067-4295 represents the peptide in the HNH domain of the above MG4 nuclease.

SEQ ID NO: 5503 shows the nucleotide sequence of MG4 tracrRNA derived from the same locus as the above MG4 nuclease.

SEQ ID NO: 5468 represent the nucleotide sequence of the sgRNA engineered to function with the MG4 nuclease.

SEQ ID NO: 5649 shows peptide sequence characteristics of MG4 family enzymes.

MG6

SEQ ID NO: 661-668 show the full length peptide sequence of MG6 nuclease.

SEQ ID NO: 2482-2489 shows the peptide sequence of the RuvC_III domain of the above MG6 nuclease.

SEQ ID NO: 4296-4303 represent peptides in the HNH domain of the above MG3 nuclease.

MG7

SEQ ID NO: 669-677 show the full length peptide sequence of MG7 nuclease.

SEQ ID NO: 2490-2498 represent the peptide sequence of the RuvC_III domain of the MG7 nuclease above.

SEQ ID NO: 4304-4212 shows the peptide of the HNH domain of the above MG3 nuclease.

SEQ ID NO: 5504 shows the nucleotide sequence of MG7 tracrRNA derived from the same locus as MG7 nuclease above.

MG14 SEQ ID NO: 678-929 shows the full length peptide sequence of MG14 nuclease.

SEQ ID NO: 2499-2750 shows the peptide sequence of the RuvC_III domain of the above MG14 nuclease.

SEQ ID NO: 4313-4564 represent peptides in the HNH domain of the above MG14 nuclease.

SEQ ID NO: 5505 shows the nucleotide sequence of MG14 tracrRNA derived from the same locus as the above MG14 nuclease.

SEQ ID NO: 5581 shows the nucleotide sequence of the codon-optimized coding sequence of E. coli of the MG14 family of enzymes.

SEQ ID NO: 5650-5667 show peptide sequence characteristics of MG14 family enzymes.

MG15

SEQ ID NO: 930-1092 shows the full-length peptide sequence of MG15 nuclease.

SEQ ID NO: 2751-2913 show the peptide sequence of the RuvC_III domain of the above MG15 nuclease.

SEQ ID NO: 4565-4727 represents the peptide of the HNH domain of the above MG15 nuclease.

SEQ ID NO: 5506 represents the nucleotide sequence of MG15 tracrRNA derived from the same locus as the MG15 nuclease above.

SEQ ID NO: 5470 represents the nucleotide sequence of the sgRNA engineered to function with the MG15 nuclease.

SEQ ID NO: 5582 show the nucleotide sequence of the E. coli codon-optimized coding sequence of the MG15 family of enzymes.

SEQ ID NO: 5668-5675 shows the peptide sequence characteristics of MG15 family enzymes.

MG16

SEQ ID NO: 1093-1353 show the full length peptide sequence of MG16 nuclease.

SEQ ID NO: 2914-3174 shows the peptide sequence of the RuvC_III domain of the above MG16 nuclease.

SEQ ID NO: 4728-4988 represent peptides in the HNH domain of the above MG16 nuclease.

SEQ ID NO: 5507 shows the nucleotide sequence of MG16 tracrRNA derived from the same locus as the MG3 nuclease above.

SEQ ID NO: 5471 represents the nucleotide sequence of the sgRNA engineered to function with the MG16 nuclease.

SEQ ID NO: 5583 shows the nucleotide sequence of the codon-optimized coding sequence of E. coli of the MG16 family of enzymes.

SEQ ID NO: 5676-5678 show the peptide sequence characteristics of MG16 family enzymes.

MG18

SEQ ID NO: 1354-1511 shows the full length peptide sequence of MG18 nuclease.

SEQ ID NO: 3175-3330 shows the peptide sequence of the RuvC_III domain of the above MG18 nuclease.

SEQ ID NO: 4989-5146 represent peptides in the HNH domain of the above MG18 nuclease.

SEQ ID NO: 5508 shows the nucleotide sequence of MG18 tracrRNA derived from the same locus as the above MG18 nuclease.

SEQ ID NO: 5472 represents the nucleotide sequence of the sgRNA engineered to function with the MG18 nuclease.

SEQ ID NO: 5584 shows the nucleotide sequence of the codon-optimized coding sequence of E. coli of the MG18 family of enzymes.

SEQ ID NO: 5679-5686 show peptide sequence characteristics of MG18 family enzymes.

MG21

SEQ ID NO: 1512-1655 show the full length peptide sequence of MG21 nuclease.

SEQ ID NO: 3331-3474 shows the peptide sequence of the RuvC_III domain of the above MG21 nuclease.

SEQ ID NO: 5147-5290 represent peptides in the HNH domain of the above MG21 nuclease.

SEQ ID NO: 5509 shows the nucleotide sequence of MG21 tracrRNA derived from the same locus as the above MG21 nuclease.

SEQ ID NO: 5473 represents the nucleotide sequence of the sgRNA engineered to function with the MG21 nuclease.

SEQ ID NO: 5585 shows the nucleotide sequence of the codon-optimized coding sequence of E. coli of the MG21 family of enzymes.

SEQ ID NOs: 5687-5692 and 5674-5675 show peptide sequence characteristics of MG21 family enzymes.

MG22

SEQ ID NO: 1656-1755 shows the full length peptide sequence of MG22 nuclease.

SEQ ID NO: 3475-3568 show the peptide sequence of the RuvC_III domain of the above MG22 nuclease.

SEQ ID NO: 5291-5389 represent peptides in the HNH domain of the above MG22 nuclease.

SEQ ID NO: 5510 shows the nucleotide sequence of MG22 tracrRNA derived from the same locus as the above MG22 nuclease.

SEQ ID NO: 5474 represents the nucleotide sequence of the sgRNA engineered to function with the MG22 nuclease.

SEQ ID NO: 5586 shows the nucleotide sequence of the E. coli codon-optimized coding sequence of the MG22 family of enzymes.

SEQ ID NO: 5694-5699 show the peptide sequence characteristics of the MG22 family of enzymes.

MG23

SEQ ID NO: 1756-1826 shows the full length peptide sequence of MG23 nuclease.

SEQ ID NO: 3569-3637 shows the peptide sequence of the RuvC_III domain of the above MG23 nuclease.

SEQ ID NO: 5390-5460 represent peptides in the HNH domain of the above MG23 nuclease.

SEQ ID NO: 5511 shows the nucleotide sequence of MG23 tracrRNA derived from the same locus as the above MG23 nuclease.

SEQ ID NO: 5475 represents the nucleotide sequence of the sgRNA engineered to function with the MG23 nuclease.

SEQ ID NO: 5587 shows the nucleotide sequence of the codon-optimized coding sequence of E. coli of the MG23 family of enzymes.

SEQ ID NO: 5700-5717 show the peptide sequence characteristics of the MG23 family of enzymes.

Although various embodiments of the invention are 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 modifications, modifications, and replacements can be conceived by one of ordinary skill in the art without departing from the present invention. It should be understood that various alternatives of the embodiments of the invention described herein may be utilized.

Implementations of some of the methods disclosed herein include immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA techniques, unless otherwise specified. Use. For example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular System (F. ), PCR 2: A Practical Aproch (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 (see R. Freshney, 20th Incorporated in the statement).

As used herein, the singular forms "one (a)", "one (an)", and "the" unless the context clearly indicates other meanings. It is also intended to include the plural. In addition, the terms "inclusion", "includes", "having", "has", "with", or variants thereof. To the extent used in any of the details and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising". There is.

The term "about" or "approximately" means that a particular value is within the permissible margin of error as determined by one of ordinary skill in the art, and that error range is how the value is measured or measured. It is determined, that is, it depends in part on the limitations of the measurement system. For example, "about" can mean a standard deviation of 1 or more than 1 per practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of any value.

As used herein, "cell" generally refers to a living cell. A cell can be a basic structural unit, a functional unit, and / or a biological unit of an organism. Cells may originate from any organism that has one or more cells. Some non-limiting examples are prokaryotic cells, eukaryotic cells, bacterial cells, paleobacterial cells, single-cell eukaryotic cells, protozoan cells, plant cells (eg, crops, fruits, vegetables). , Grain, soybean, corn, corn, corn, wheat, seed, tomato, rice, cassaba, sugar cane, pumpkin, hay, potato, cotton, asa, tobacco, flowering plant, conifer, nude plant, fern, Hikagenokazura, Tsunogoke, Moss, 蘚 cells), Algae cells (eg, Botryococcus braunii, Chlamydomonas reinhardti, Nannochlopsis gaditana, Chlorella pyre), etc. , Yeast cells, cells from mushrooms), animal cells, spinal cord animals (eg, gypsum flies, spores, thorns, nematodes, etc.), vertebrates (eg, fish, amphibians, reptiles, birds, mammals, etc.) Examples include cells of animals), cells of mammals (eg, pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not have their origin in natural organisms (eg, cells are synthetically produced and are sometimes called artificial cells).

The term "nucleotide", as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (eg, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide includes ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphate. For example, dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof may be included. Such derivatives may include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on nucleic acid molecules containing them. The term nucleotide can refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives, as used herein. Exemplary examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or labeled for detection, such as by using moieties that contain optically detectable moieties (eg, fluorophores). 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-carboxyfluorosein (JOE), rhodamine, 6-carboxyrhodamine. (R6G), N, N, N', N'-Tetramethyl-6-Rhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 4- (4'dimethylaminophenylazo) benzoic acid (DABCYL) ), Cascade Blue, Fluorescein, 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, available from Perkin Elmer (Foster City, Calif). JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] d Amersham (Arlington Heights, Ill) available from a FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP; Boehringer Mannheim (Indianapolis, Ind.) from Available fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'- dATP; and Chromosome Labeled Nucleotidines, BODICY-FL-14-UTP, BODICY-FL-4-UTP, BODICY-TMR-14-UTP, BODICY-TM available from Molecular Probes (Eugene, Oreg). , BODICY-TR-14-UTP, BODICY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluolecein-12-UTP, Fluolecein-12-dUTP, Oregon Green 488-5-dUTP , Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethyl Rhodamine 6-UTP, Tetramethyl Rhodamine 6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP. Listed Can be. Nucleotides can also be labeled or marked by chemical modification. The single chemically modified nucleotide may be biotin dNTP. Some non-limiting examples of biotinylated dNTPs include bio-dATP (eg, bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (eg, biotin-11-dCTP, biotin-). 14-dCTP), and biotin-dUTP (eg, biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) can be mentioned.

The terms "polynucleotide", "oligonucleotide", and "nucleic acid" generally refer to nucleotides of any length, either in the form of single-stranded, double-stranded, or multi-stranded. Used interchangeably to refer to the high molecular weight form of (either deoxyribonucleotide or ribonucleotide), or its analogs. Polynucleotides can be exogenous or endogenous to cells. The polynucleotide may be a gene or fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may be optional. It may also have a three-dimensional structure of, and may perform any function. A polynucleotide may contain one or more analogs (eg, a modified skeleton, sugar, or nucleobase). Modifications to the nucleotide structure, if present, can be given before or after assembly of the polymer. Some non-limiting examples of analogs are 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked. Nucleic Acids, Glycol Nucleic Acids, Treose Nucleic Acids, Dideoxynucleotides, Cordisepines, 7-Daza-GTP, Fluorophores (eg, Sugar-Binding Rhodamine or Fluolecein), Nucleotide-Containing Thiols, Biotin-Binding Nucleotides, Fluorescent Base Analogs ), CpG islands, methyl-7-guanosine, methylated nucleotides, inosin, thiouridine, pseudourdine, dihydrouridine, cuosin, and wyosine. Non-limiting examples of polynucleotides include genes or genes. Coding or non-coding regions of the fragment, nucleotides defined from linkage analysis, exons, introns, messenger RNAs (mRNAs), translocated RNAs (tRNAs), ribosome RNAs (rRNAs), small interfering RNAs (siRNAs), small molecules Hairpin-type RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, Cell-free polynucleoti containing 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.

The term "transfected" or "transfected" generally refers to the introduction of nucleic acid into a cell by a non-virus-based method or a virus-based method. A nucleic acid molecule can be a complete protein or a gene sequence encoding a functional portion thereof. For example, Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by a peptide bond. The term does not imply a particular length of polymer and also implies or identifies whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. Not intended. 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, as well as proteins with or without secondary and / or tertiary structures (eg, domains). The term also includes amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, oxidation, and other operations, eg, conjugates with labeled components. The term "amino acid", as used herein, generally refers to natural amino acids and unnatural amino acids, including modified amino acids and amino acid analogs. Modified amino acids may include natural and unnatural amino acids, which are chemically modified to include non-naturally occurring groups or chemical moieties on the amino acids. Amino acid analogs may also refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

As used herein, "non-natural" generally refers to nucleic acid or polypeptide sequences not found in natural nucleic acids or proteins. Non-natural may refer to affinity tags. Non-natural may refer to fusion. Non-natural may refer to a naturally occurring nucleic acid or polypeptide sequence containing mutations, insertions, and / or deletions. Non-natural sequences include activities that may be exhibited by nucleic acid and / or polypeptide sequences to which the non-natural sequences are fused (eg, enzyme activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc. ) May be indicated and / or coded. Non-natural nucleic acid or polypeptide sequences are genetically engineered to bind to naturally occurring nucleic acid or polypeptide sequences (or variants thereof) and encode chimeric nucleic acids and / or chimeric nucleic acids and / or polypeptides. May produce a polypeptide sequence.

As used herein, the term "promoter" generally refers to a regulatory DNA region that controls transcription or expression of a gene, adjacent to or overlapping a nucleotide or region of nucleotide in which RNA transcription is initiated. May be located. Promoters may contain specific DNA sequences that bind to protein factors, often referred to as transcription factors, which facilitate the binding of RNA polymerase to DNA and trigger gene transcription. A "basic promoter," also referred to as a "core promoter," generally refers to a promoter that contains all the basic elements necessary to promote transcriptional expression of operably linked polynucleotides. Eukaryotic basal promoters typically, but not necessarily, contain TATA boxes and / or CAAT boxes.

The term "expression", as used herein, generally refers to the process by which a nucleic acid sequence or polynucleotide is transcribed (such as mRNA or other RNA transcript) from a DNA template and / or transcription. Refers to the process by which the resulting mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides are sometimes collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

As used herein, "operably concatenated", "operable concatenation", or "operably concatenated", or grammatical equivalents thereof, are generally genetic elements, eg, eg. It refers to juxtaposition of promoters, enhancers, polyadenylation sequences, etc., and these elements are in a relationship that allows them to operate in the expected manner. For example, a regulatory element that may include a promoter and / or enhancer sequence is operably linked to the coding region if it assists the regulatory element in initiating transcription of the coding sequence. As long as this functional relationship is maintained, there may be residues intervening between the regulatory element and the coding region.

As used herein, "vector" generally refers to a macromolecule or macromolecular assembly that contains or associates with a polynucleotide, which is a polynucleotide to a cell. Can be used to mediate delivery. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. Vectors generally include genetic elements operably linked to the gene to promote expression of the gene in the target, eg, regulatory elements.

As used herein, "expression cassette" and "nucleic acid cassette" are generally exchanged to refer to a combination of nucleic acid sequences or elements that are expressed together or operably linked for expression. Used as possible. In some cases, an expression cassette refers to a combination of regulatory elements and genes to which they are operably linked for expression.

A "functional fragment" of a DNA or protein sequence generally refers to a fragment that retains a biological activity (functional or structural) that is substantially similar to the biological activity of a full-length DNA or protein sequence. The biological activity of a DNA sequence can be its ability to influence expression in a manner known to be due to the full-length sequence.

As used herein, a "manipulated" subject generally indicates that the subject has been modified by human intervention. According to a non-limiting example, a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; the nucleic acid possesses a function that the ligated product does not exist in the original nucleic acid. As such, the nucleic acid may be modified by ligating the nucleic acid to a nucleic acid that the nucleic acid does not associate with in nature; the engineered nucleic acid may be synthesized in vitro with a sequence that does not exist in nature; May be modified by changing its amino acid sequence to a sequence that does not exist in nature; the engineered protein may gain new functions or properties. An "operated" system comprises at least one manipulated component.

As used herein, "synthetic" and "artificial" have low sequence identity (eg, less than 50% sequence identity, less than 25% sequence identity) with respect to naturally occurring human proteins. Used interchangeably to refer to a protein or domain thereof having less than 10% sequence identity, less than 5% sequence identity, less than 1% sequence identity). For example, the VPR and VP64 domains are synthetic transactivation domains.

The term "tracrRNA" or "tracr sequence", as used herein, is generally a wild-type exemplary tracrRNA sequence (eg, tracrRNA, or sequence from S. pyogenes, Staphylococcus aureus, etc.). Number: 5476-5511) with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identical. It may refer to a nucleic acid having sex and / or a sequence similar to the exemplary tracrRNA sequence of its wild form. TracrRNA is up to about 5%, 10%, 20%, 30%, 40%, 50%, relative to wild-type exemplary tracrRNA sequences (eg, tracrRNA from S. pyogenes, Staphylococcus aureus, etc.). It may refer to a nucleic acid having 60%, 70%, 80%, 90%, or 100% sequence identity and / or a sequence similar to its wild-type exemplary tracrRNA sequence. TracrRNA may refer to a modified form of tracrRNA, including nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeras. When a tracrRNA refers to a nucleic acid that is at least about 60% identical to a wild-type exemplary tracrRNA (eg, tracrRNA from S. pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least 6 consecutive nucleotides. There is. For example, the tracrRNA sequence is at least about 60% identical to, at least about 65, the wild-type exemplary tracrRNA (eg, tracrRNA from S. pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least 6 consecutive nucleotides. % 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%, at least about 99% identical, or 100% identical. Type II tracrRNA sequences can be predicted on genomic sequences by identifying regions of complementarity to some of the repetitive sequences in adjacent CRISPR arrays.

As used herein, "guide nucleic acid" may generally refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid may be RNA. The guide nucleic acid may be DNA. The guide nucleic acid may be programmed to bind site-specifically to the sequence of nucleic acids. The targeted nucleic acid or target nucleic acid may contain nucleotides. The guide nucleic acid may contain nucleotides. The portion of the target nucleic acid may be complementary to the portion of the guide nucleic acid. The strand of the double-stranded target polynucleotide that is complementary to the guide nucleic acid and hybridizes to the guide nucleic acid is sometimes referred to as the complementary strand. The strands of a double-stranded target polynucleotide that are complementary to the complementary strand and therefore may not be complementary to the guide nucleic acid are sometimes referred to as noncomplementary strands. The guide nucleic acid may contain one polynucleotide chain and may be referred to as a "single guide nucleic acid". The guide nucleic acid may contain two polynucleotide chains and may be referred to as a "double guide nucleic acid". Unless otherwise stated, the term "guide nucleic acid" is comprehensive and may refer to both single and double guide nucleic acids. The guide nucleic acid may include a segment, which may be referred to as a "nucleic acid targeting segment" or a "nucleic acid targeting sequence". Nucleic acid targeting segments may include subsegments that may be referred to as "protein binding segments" or "protein binding sequences" or "Cas protein binding segments".

In the context of two or more nucleic acid or polypeptide sequences, the term "sequence identity" or "percent identity" generally refers to two (eg, pairwise alignment) or more (eg, multiple sequence alignment). Refers to sequences, which are the same or the same when compared or aligned for maximum correspondence across the local or overall comparison window, as measured using the sequence comparison algorithm. Has the same amino acid residue or specified proportion of nucleotides. Suitable sequence comparison algorithms for polypeptide sequences use, for example, BLASTUM62 scoring matrix parameters that set the gap cost at 3 wordsth (W), 10 exposure (E), and 11 extensions and 1 extensions. And using BLASTP using conditional compositional score matrix algorithm for polypeptide sequences longer than 30 residues; 2 wordlength (W), 10000 extraction (E) parameters, and 30. BLASTP using a PAM30 scoring matrix that sets the gap cost at 9 to open the gap and 1 to extend the gap for sequences less than residues (these are https://blast.ncbi.nlm). It is the default parameter of BLASTP in BLAST suite available in .nih.gov); CLASTALW with parameters; Smith-Waterman homology search algorithm with parameters of 2 match, -1 mismatch, and -1 gap; Includes MUSCLE with default parameters; MAFFT with 2 regions and 1000 maxiterations parameters; Novafold with default parameters; HMMER hmmalign with default parameters.

As used herein, the term "RuvC_III domain" is generally three of the RuvC endonuclease domain (the RuvC nuclease domain composed of the three discontinuous segments RuvC_I, RuvC_II, and RuvC_III). Refers to the discontinuous segment of the eye. A RuvC domain or segment thereof is generally a Hidden Markov Model (HMM) constructed by alignment to a known domain sequence, structural alignment to a protein having an annotated domain, or based on a known domain sequence (eg, in RuvC_III). It can be identified by comparison with Pfam HMM PF18541).

As used herein, the term "HNH domain" generally refers to an endonuclease domain with characteristic histidine and asparagine residues. HNH domains are generally Hidden Markov Models (HMMs) constructed by alignment to known domain sequences, structural alignments to proteins with annotated domains, or based on known domain sequences (eg, Pfam HMM in domain HNH). It can be identified by comparison with PF01844).

Overview

The discovery of a new Cas enzyme with a unique function and structure presents the potential to further disrupt deoxyribonucleic acid (DNA) editing techniques, improving speed, specificity, functionality, and ease of use. Can be done. CRISPR / functionally characterized in comparison to the predicted prevalence and diverse microbial species of clustered and regularly arranged short palindromic sequence repeat (CRISPR) systems in microorganisms. Cas enzymes are relatively rare in the literature. This is partly because huge numbers of microbial species may not be easily cultivated in laboratory conditions. Metagenomic sequencing from natural environmental niches representing many microbial species may suggest that the number of known new CRISPR / Cas systems may increase exponentially and facilitate the discovery of new oligonucleotide editing functions. A recent example of the benefits of such an approach is demonstrated by the 2016 discovery of the CasX / CasY CRISPR system from metagenomic analysis of natural microorganisms.

The CRISPR / Cas system is an RNA-directed nuclease complex described to function as an adaptive immune system in microorganisms. In their natural context, a CRISPR / Cas system occurs at a CRISPR (clustered, regularly arranged short palindromic sequence repeat) operon or locus, which generally has two parts: (i) RNA-based targeting. An array of short repeats (30-40bp) separated by equally short spacer sequences encoding the elements; (ii) encoding CRISPR polypeptides directed by RNA-based targeting elements, along with accessory proteins / accessory enzymes. Includes an ORF that encodes Cas. Efficient nuclease targeting of a particular target nucleic acid sequence generally involves (i) complementary hybridization between the first 6-8 nucleic acids of the target (target seeded) and the crRNA guide; ii) It requires both the presence of a protospacer-adjacent motif (PAM) sequence within the defined neighborhood of the target seed (PAM is generally a sequence that is not commonly represented within the host genome). Depending on the exact function and configuration of the system, the CRISPR-Cas system will generally be divided into two classes, five types, and 16 subtypes, based on shared functional characteristics and evolutionary similarities. Be organized.

Class I CRISPR-Cas systems have large multi-subunit effector complexes, including type I, type III, and type IV.

Type I CRISPR-Cas systems are considered to be of moderate complexity in terms of components. In the CRISPR-Cas system of type I, an array of elements targeting RNA is transcribed as a long precursor crRNA (pre-crRNA), which is processed with repetitive elements to release the short mature crRNA, which is short and mature. The resulting crRNAs are followed by a suitable short consensus sequence called a protospacer flanking motif (PAM), which directs the nuclease complex to the nucleic acid target. This treatment is carried out via the endoribonuclease subunit (Cas6) of a large endonuclease complex called cascade, which further comprises the nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nuclease mainly functions as a DNA nuclease.

The CRISPR system of type III may be characterized by the presence of a central nuclease known as Cas10, along with a repeat-associated mysterious protein (RAMP) containing a protein subunit of Csm or Cmr. Like the type I system, mature crRNA is processed from pre-crRNA using an enzyme such as Cas6. Unlike the Type I and Type II systems, the Type III system appears to target and cleave DNA-RNA duplexes, such as the DNA strand used as a template for RNA polymerase.

The type IV CRISPR-Cas system consists of two genes, a highly reduced large subunit nuclease (csf1) and a plasmid protein in the Cas5 (csf3) and Cas7 (csf2) groups, and in some cases, the RAMP protein. Has an effector complex consisting of one gene of the predicted small subunit; such systems are commonly found on endogenous plasmids.

Class II CRISPR-Cas systems generally have a single polypeptide multidomain nuclease effector, including type II, type V, and type VI.

The type II CRISPR-Cas system is considered to be the simplest in terms of components. In the type II CRISPR-Cas system, processing a CRISPR array into a mature crRNA is not a small transcoded crRNA (tracrRNA) with regions complementary to the array repeats, but a special one. The presence of an endonuclease subsystem is required; tracrRNA interacts with both its corresponding effector nuclease (eg Cas9) and repetitive sequences to form a precursor dsRNA structure, which is an intrinsic cause. It is cleaved by sex RNAse III to produce 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 employs an RNase H fold in which an irrelevant HNH nuclease domain is inserted within the fold of the RuvC-like nuclease domain. The RuvC-like domain causes cleavage of the target (eg, crRNA-complementary) DNA strand, while the HNH domain causes cleavage of the substituted DNA strand.

The V-type CRISPR-Cas system features a nuclease effector (eg, Cas12) structure similar to the nuclease effector of type II effectors, including the RuvC-like domain. Like type II, most (but not all) V-type CRISPR systems use tracrRNAs to process pre-crRNAs into mature crRNAs; however, they cleave the pre-crRNAs into multiple crRNAs. Unlike type II systems, which require RNAse III to do so, type V systems can use the effector nuclease itself to cleave precrRNA. Similar to the type II CRISPR-Cas system, the type V CRISPR-Cas system is also known as a DNA nuclease. Unlike the Type II CRISPR-Cas system, some V-type enzymes (eg, Cas12a) are robust single-stranded non-strands activated by the first crRNA-directed cleavage of the double-stranded target sequence. It appears to have specific deoxyribonuclease activity.

The VI-type CRISPR-Cas system has an RNA-guided RNA endonuclease. Instead of a RuvC-like domain, a single polypeptide effector in a VI-type system (eg, Cas13) comprises two HEPN ribovocrim domains. Unlike both type II and type V systems, the type VI system does not appear to require tracrRNA to process pre-crRNA into crRNA. However, like the V-type system, some VI-type systems (eg, C2C2) are robust single-stranded non-specific nucleases activated by the first crRNA-directed cleavage of the target RNA. Seems to have (ribonuclease) activity.

Class II CRISPR-Cas, due to its simpler structure, is most widely adopted for engineering and development as a designer nucleose / genome editing application.

One of the early adaptations of such a system for in vitro use is Jinek et al. (Science. 2012 Aug 17; 337 (6096): 816-2, which is hereby fully by reference. Can be seen in (embedded). In Jinek's test, (i) S. Full-length Cas9 (eg, Class II Type II Casenase) isolated from pyogenes SF370, recombinantly expressed and purified, (ii) a target DNA sequence desired to be cleaved. Purified mature to 42 nt crRNA with a ~ 20 nt 5'sequence complementary to, followed by a 3'tracr binding sequence (the entire crRNA is transcribed in vitro from a synthetic DNA template with the T7 promoter sequence); A system containing purified tracrRNA, transcribed in vitro from a synthetic DNA template with the iii) T7 promoter sequence, and (iv) Mg2 + was first described. Jinek then described an improved engineered system in which to form a single fused synthetic guide RNA (sgRNA) capable of targeting Cas9 on its own. The crRNA of ii) is bound to the 5'end of (iii) by a linker (eg, GAAA) (compare the upper and lower panels of FIG. 2).

Mari et al. (Science. 2013 Feb 15; 339 (6121): 823-826.), Which is fully incorporated herein by reference, are then described in (i) C-terminal nuclear localization sequences (i) For example, with an ORF encoding codon-optimized Cas9 (eg, Class II type II Cas enzyme) under a suitable mammalian promoter with SV40 NLS) and a suitable polyadenylation signal (eg, TK pA signal); ii) An ORF (starting with G, 5'sequence followed by a complementary targeting nucleic acid sequence, 20 nt, followed by a 3'tracr binding sequence bound to it, under the appropriate polymerase III promoter (eg, U6 promoter). The system was adapted for use in mammalian cells by providing a DNA vector encoding (with a linker and a promoter RNA sequence).

MG1 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NO: 1827-2140. In some cases, the endonuclease may contain a RuvC_III domain, wherein the RuvC_III domain is described in SEQ ID NO: 1827-2140. 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 about any one of them. 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 about It has 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity. In some cases, endonucleases may contain the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 1827-2140. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 1827-1831. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45 relative to any one of SEQ ID NOs: 1827-1831. %, 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%, including RuvC_III domains having at least about 99% identity. In some cases. In some cases, endonucleases may contain the RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 1827-1831. In some cases, endonucleases are 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% relative to SEQ ID NO: 1827. May include a RuvC_III domain having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity. In some cases, endonucleases are 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%, relative to SEQ ID NO: 1828. It may contain a RuvC_III domain having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity. In some cases, endonucleases are 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%, relative to SEQ ID NO: 1829. It may contain a RuvC_III domain having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity. In some cases, endonucleases are 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%, relative to SEQ ID NO: 1830. It may contain a RuvC_III domain having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity. In some cases, endonucleases are 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%, relative to SEQ ID NO: 1831. It may contain a RuvC_III domain having at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identity.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3955. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 relative to any one of SEQ ID NOs: 3638-3955. %, 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% HNH domains with identity. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 3638-3955. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3955. In some cases, the endonuclease is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 relative to any one of SEQ ID NOs: 3638-3955. %, 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% HNH domains with identity. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 3638-3955. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3461. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 relative to any one of SEQ ID NOs: 3638-3461. %, 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% HNH domains with identity. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 3638-3461. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NO: 3638. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, relative to any one of SEQ ID NO: 3638. When containing an HNH domain having 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. There is. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NO: 3638. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NO: 3639. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, relative to any one of SEQ ID NO: 3639. When containing an HNH domain having 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. There is. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NO: 3639. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NO: 3640. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, relative to any one of SEQ ID NO: 3640. When containing an HNH domain having 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. There is. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NO: 3640. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NO: 3641. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, relative to any one of SEQ ID NO: 3641. When containing an HNH domain having 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. There is. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NO: 3641.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% with respect to any one of SEQ ID NOs: 1-6 or 9-319. , 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% May contain variants having at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1-6 or 9-319. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, and at least about 55 relative to any one of SEQ ID NOs: 1-4. %, 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% of variants may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1-4. In some cases, endonucleases may contain a peptide motif that is substantially identical to any one of SEQ ID NOs: 5615, 5616, or 5617.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS can be proximal to the N-terminus or C-terminus of the endonuclease. NLS can be added to the N-terminus or C-terminus of any one of SEQ ID NOs: 1-6 or 9-319, or at least about 30% of any one of SEQ ID NOs: 1-319. 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%, Addition to the N-terminus or C-terminus of a variant having 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 Can be done. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. When the NLS comprises 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: 5593-5608. There is. The NLS may contain a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS may include any or a combination of the sequences in Table 1 below.

場合によっては、エンドヌクレアーゼは、組換え型であり得る（例えば、大腸菌中の発現とそれに続くエピトープタグ精製などの適切な方法によって、クローン化され、発現され、および精製される）。場合によっては、エンドヌクレアーゼは、配列番号：５５９２－５５９５のいずれか１つに対して少なくとも約９０％の同一性を有する１６Ｓ ｒＲＮＡ遺伝子を有する細菌に由来する場合がある。エンドヌクレアーゼは、配列番号：５５９２－５５９５のいずれか１つと少なくとも約８０％、少なくとも約８２％、少なくとも約８３％、少なくとも約８４％、少なくとも約８５％、少なくとも約８６％、少なくとも約８７％、少なくとも約８８％、少なくとも約８９％、少なくとも約９０％、少なくとも約９１％、少なくとも約９２％、少なくとも約９３％、少なくとも約９４％、少なくとも約９５％、少なくとも約９６％、少なくとも約９７％、少なくとも約９８％、または少なくとも約９９％同一の１６Ｓ ｒＲＮＡ遺伝子を有する種に由来する場合がある。エンドヌクレアーゼは、配列番号：５５９２－５５９５のいずれか１つと実質的に同一の１６Ｓ ｒＲＮＡ遺伝子を有する種に由来する場合がある。エンドヌクレアーゼは、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａ門またはＣａｎｄｉｄａｔｕｓ Ｐｅｒｅｇｒｉｎｉｂａｃｔｅｒｉａ門に属する細菌に由来する場合がある。

In some cases, endonucleases can be recombinant (eg, cloned, expressed, and purified by appropriate methods such as expression in E. coli followed by epitope tag purification). In some cases, the endonuclease may be derived from a bacterium having a 16S rRNA gene with at least about 90% identity to any one of SEQ ID NOs: 5592-5595. Endonucleases are at least about 80%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, with any one of SEQ ID NOs: 5592-5595. 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%, It may be derived from a species that has at least about 98%, or at least about 99%, the same 16S rRNA gene. The endonuclease may be derived from a species having a 16S rRNA gene that is substantially identical to any one of SEQ ID NOs: 5592-5595. Endonucleases may be derived from bacteria belonging to the phylum Verrucomicrobiota or the phylum Candidatus Peregrinibacteria.

In some cases, sequence identity can be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity is determined using the BLASTUM62 scoring matrix, which sets the gap cost using the parameters of 3 wordlength (W), 10 extraction (E), and 11 exhibitions, 1 extension, and conditions. It may be determined by the BLASTP algorithm using a combined composition score matrix adjustment.

In some cases, the system described above may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). ) May be included. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, the nucleotide on the 5'most side of the targeting region (5'most nucleotide) may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence can be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA can include from 5'to 3': an unnatural guide nucleic acid sequence capable of hybridizing to a target sequence in the cell; and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a particular sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). With respect to at least about 80%. The tracr sequence is at least about 60-100 of any one of SEQ ID NOs: 5476-5489 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about about. It may have at least about 80% sequence identity to the contiguous nucleotides of 90). In some cases, the tracrRNA is at least about 60-90 of any one of SEQ ID NOs: 5476-5489 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Or 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 about 90) contiguous nucleotides. They may have 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the tracrRNA is at least about 60-100 of any one of SEQ ID NOs: 5476-5489 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Or it may be substantially identical to at least about 90) contiguous nucleotides. The tracrRNA may contain any of SEQ ID NOs: 5476-5489.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease is at least about 80% identical to any one of SEQ ID NOs: 5461-5464. May contain sequences of sex. The sgRNA is 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% with respect to any one of SEQ ID NOs: 5461-5464. %, At least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of sequences having the same identity. The sgRNA may contain a sequence that is substantially identical to any one of SEQ ID NOs: 5461-5464.

In some cases, the above system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second site is the second. It is 3'of 1 part. In some cases, the above system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) in 5'of the first region, from 5'to 3'. A first homology arm containing a sequence of nucleotides, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 3'in the second region. It may contain a single-stranded or double-stranded DNA repair template, including a second homology arm containing a sequence of 300, 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a target nucleic acid locus. The method comprises delivering any of the unnatural systems disclosed herein to a target nucleic acid locus, comprising an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein. Can include. The enzyme forms a complex with at least one sgRNA, and when the complex binds to the target nucleic acid locus, it may modify the target nucleic acid locus. Delivering an enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering a nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering a nuclease to the locus may include incubating the nucleic acid containing the locus of interest with the system in buffer. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic or prokaryotic cells. The cell may be an animal cell, a human cell, a bacterial cell, a paleobacterial cell, or a plant cell. Enzymes can cause single-strand or double-strand breaks at or proximal to the target locus of interest.

If the target nucleic acid locus may be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NO: 1827-2140. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease is at least about 30 relative to any one of SEQ ID NO: 5571-5575 or any one of SEQ ID NO: 5571-5575. %, 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% of the variant having the same identity. Can contain an array of. In some cases, the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be supplied as a translated polypeptide. Even if the at least one engineered sgRNA is supplied as a deoxyribonucleic acid (DNA) containing a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. good. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

In some cases, the disclosure may provide an expression cassette comprising the systems disclosed herein, or the nucleic acids described herein. In some cases, the expression cassette or nucleic acid may be supplied as a vector. In some cases, the expression cassette, nucleic acid, or vector may be supplied intracellularly. In some cases, the cell is a bacterial cell having a 16S rRNA gene with at least about 90% (eg, at least about 99%) identity to any one of SEQ ID NOs: 5592-5595.

MG2 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2141-2241. In some cases, the endonuclease may contain a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30% with respect to any one of SEQ ID NOs: 2141-2241. 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%. , Have at least about 99% identity. In some cases, the endonuclease may contain the RuvC_III domain and is substantially identical to any one of SEQ ID NOs: 2141-2142. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2141-2142. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45 relative to any one of SEQ ID NOs: 2141-2142. %, 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 RuvC_III domain having an identity of 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%. May include. In some cases, endonucleases may contain the RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2141-2142.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955-4055. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 relative to any one of SEQ ID NOs: 3955-4055. %, 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% if it contains HNH domains that are identical. There is. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 3955-4055. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955-3956. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least for any one of SEQ ID NOs: 3955-3956. HNH domains that are about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 3955-3956.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 320-420. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-420. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 320-321. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-321.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 for any one of SEQ ID NOs: 320-420 at the N-terminus or C-terminus, or for any one of SEQ ID NOs: 320-420. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is at least about 60-100 of any one of SEQ ID NOs: 5490-5494 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about about. It may have at least about 80% sequence identity to the contiguous nucleotides of 90). In some cases, the tracrRNA is at least about 60-90 of any one of SEQ ID NOs: 5490-5494 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Or 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 about 90) contiguous nucleotides. It may have 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the tracrRNA is at least about 60-100 of any one of SEQ ID NOs: 5490-5494 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Alternatively, it may be substantially identical to at least about 90) contiguous nucleotides. The tracrRNA may comprise any of SEQ ID NOs: 5490-5494.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5465. .. The sgRNA is 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%, relative to SEQ ID NO: 5465. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5465.

In some embodiments, the system may include two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region. Is 3'for the first region. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2141-2241. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially the same as any one of SEQ ID NO: 5576-5571, or any one of SEQ ID NO: 5576-5571. 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% of variants having identity. It may be included. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG3 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2242-2251. In some cases, the endonuclease may include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2242-2251. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2242-2251. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2242-2244. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2242-2244. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2242-2244.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056-4066. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4056-4066. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4056-4066. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056-4058. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4056-4058. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4056-4058.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 421-431. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-431. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 421-423. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-423.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 421-431 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 421-431. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is at least about 60-100 of any one of SEQ ID NOs: 5495-5502 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about about. It may have at least about 80% sequence identity to the contiguous nucleotides of 90). In some cases, the tracrRNA is at least about 60-90 of any one of SEQ ID NOs: 5495-5502 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Or 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 about 90) contiguous nucleotides. It may have 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the tracrRNA is at least about 60-100 of any one of SEQ ID NOs: 5495-5502 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, Alternatively, it may be substantially identical to at least about 90) contiguous nucleotides. The tracrRNA may comprise any of SEQ ID NOs: 5495-5502.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease has at least about 80% identity to any one of SEQ ID NOs: 5466-5467. May include sequences having. The sgRNA is 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% with respect to any one of SEQ ID NOs: 5466-5467. %, At least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of sequences having the same identity. The sgRNA may contain a sequence that is substantially identical to any one of SEQ ID NOs: 5466-5467.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2242-2251. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially the same as any one of SEQ ID NO: 5578-5580, or any one of SEQ ID NO: 5578-5580. 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% of variants having identity. It may be included. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG4 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NO: 2253-2481. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NO: 2253-2481. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2253-2481. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2253-2481. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2253-2481. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2253-2481.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067-4295. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4067-4295. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4067-4295. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067-4295. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4067-4295. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4067-4295.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 432-660. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 432-660. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 432-660 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 432-660. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5503 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5503 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5503 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5503.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5468. .. The sgRNA is 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%, relative to SEQ ID NO: 5468. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5468.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NO: 2253-2481. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG6 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NO: 2482-2489. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2482-2489. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2482-2489.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4296-4303. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4296-4303. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4056-4066.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 661-668. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 661-668.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 661-668 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 661-668. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to.

In some cases, the system may contain two different guide RNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is. 3'for the first region. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NO: 2482-2489. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG7 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2490-2498. In some cases, the endonuclease may include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2490-2498. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2490-2498. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2490-2498. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2490-2498. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2490-2498.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304-4212. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4304-4212. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4304-4212. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304-4212. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4304-4212. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4304-4212.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 669-677. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 669-677. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 669-677 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 669-677. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5504 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5504 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5504 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5504.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2490-2498. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG14 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2499-2750. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2499-2750. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2499-2750. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2499-2750. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2499-2750. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2499-2750.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313-4564. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4313-4564. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4313-4564. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313-4564. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4067-4295. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4313-4564.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 678-929. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 678-929. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 678-929 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 678-929. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5505 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5505 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5505 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5505.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5469. .. The sgRNA is 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%, relative to SEQ ID NO: 5469. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5469.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2499-2750. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5581, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5581. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG15 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2751-2913. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2751-2913. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2751-2913. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2751-2913. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2751-2913. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2751-2913.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565-4727. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4565-4727. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4565-4727. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565-4727. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4565-4727. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4565-4727.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 930-1092. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 930-1092. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 930-1092 at the N-terminus or C-terminus, or to any one of SEQ ID NOs: 930-1092. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5506 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5506 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5506 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5506.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5470. .. The sgRNA is 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%, relative to SEQ ID NO: 5470. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5470.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2751-2913. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5582, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5582. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG16 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2914-3174. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 2914-3174. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 2914-3174. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2914-3174. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 2914-3174. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2914-3174.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728-4988. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4728-4988. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4728-4988. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728-4988. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4728-4988. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4728-4988.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1093-1353. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1093-1353. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at the N-terminus or C-terminus to any one of SEQ ID NO: 1093-1353, or at least about 30%, at least about 35%, to any one of SEQ ID NO: 1093-1353. 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%, It may be added to a variant having 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. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5507 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5507 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5507 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5507.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5471. .. The sgRNA is 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%, relative to SEQ ID NO: 5471. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence substantially identical to SEQ ID NO: 5471.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 2914-3174. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5583, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5583. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG18 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3175-3300. In some cases, the endonuclease may include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 3175-3300. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 3175-3300. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3175-3300. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 3175-3300. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3175-3300.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989-5146. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4989-5146. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4989-5146. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989-5146. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 4989-5146. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 4989-5146.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1354-1511. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1354-1511. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 1354-1511 at the N-terminus or C-terminus, or to any one of SEQ ID NO: 1354-1511. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5508 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5508 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5508 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5508.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5472. .. The sgRNA is 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%, relative to SEQ ID NO: 5472. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5472.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 3175-3300. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5584, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5584. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG21 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3331-3474. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 3331-3474. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 3331-3474. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3331-3474. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 3331-3474. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3331-3474.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147-5290. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5147-5290. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5147-5290. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147-5290. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5147-5290. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5147-5290.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1512-1655. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1512-1655. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 1512-1655 at the N-terminus or C-terminus, or to any one of SEQ ID NOs: 1512-1655. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5509 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5509 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5509 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5509.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5473. .. The sgRNA is 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%, relative to SEQ ID NO: 5473, It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5473.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 3331-3474. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5585, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5585. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG22 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3475-3568. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 3475-3568. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 3475-3568. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3475-3568. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 3475-3568. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3475-3568.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291-5389. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5291-5389. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5291-5389. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291-5389. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5291-5389. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5291-5389.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1656-1755. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1656-1755. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 432-660 at the N-terminus or C-terminus, or to any one of SEQ ID NOs: 1656-1755. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5510 of at least about 60-100 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5510 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5510 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5510.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5474. .. The sgRNA is 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%, relative to SEQ ID NO: 5474. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5474.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 3475-3568. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5586, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5586. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

MG23 enzyme

In one aspect, the present disclosure provides an engineered nuclease system comprising (a) endonucleases. 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 include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 3569-3637. In some cases, the endonuclease may comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least for any one of SEQ ID NOs: 3569-3637. 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 It has about 98%, at least about 99% identity. In some cases, the endonuclease may include the RuvC_III domain, which is substantially identical to any one of SEQ ID NOs: 3569-3637. The endonuclease may contain a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3569-3637. In some cases, endonucleases are at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about about any one of SEQ ID NOs: 3569-3637. 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 about RuvC_III domains with 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%, and at least about 99% identity. It may be included. In some cases, the endonuclease may contain a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3569-3637.

The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390-5640. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5390-5460. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5390-5460. The endonuclease may contain an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390-5460. In some cases, endonucleases are at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about about any one of SEQ ID NOs: 5390-5460. HNH domains with 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. May include. The endonuclease may contain an HNH domain that is substantially identical to any one of SEQ ID NOs: 5390-5460.

In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1756-1826. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826. In some cases, endonucleases are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about about any one of SEQ ID NOs: 1756-1826. 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 about It may contain variants having 97%, at least about 98%, or at least about 99% identity. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826.

In some cases, endonucleases may contain variants with one or more nuclear localization sequences (NLS). The NLS may be proximal to the N-terminus or C-terminus of the endonuclease. NLS is at least about 30%, at least about 35%, at least about 40 to any one of SEQ ID NOs: 1756-1826 at the N-terminus or C-terminus, or to any one of SEQ ID NOs: 1756-1826. %, 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% may be added at the N-terminus or C-terminus of the variant. The NLS may be the SV40 large T antigen NLS. The NLS may be a c-myc NLS. The NLS comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% identity to any one of SEQ ID NOs: 5593-5608. Can be done. The NLS can comprise a sequence that is substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any or a combination of the sequences in Table 1.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity uses the BLASTUM62 scoring matrix, which uses parameters of 3 wordlength (W), 10 extraction (E), and sets the gap cost at 11 exhibitions, 1 extension, and conditional composition. Score matrix adjustments can be used to determine with the BLASTP algorithm.

In some cases, the system may (b) form a complex with an endonuclease having a 5'targeting region complementary to the desired cleavage sequence, at least one engineered synthetic guide ribonucleic acid (sgRNA). May include. In some cases, the 5'targeting region may contain PAM sequences compatible with endonucleases. In some cases, most of the 5'nucleotides in the targeting region may be G. In some cases, the 5'targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be supplied as separate ribonucleic acid (RNA) or single ribonucleic acid (RNA). The guide RNA may contain a crRNA tracrRNA binding sequence in 3'of the targeting region. The guide RNA may contain a tracrRNA sequence in which the 4-nucleotide linker precedes the 3'of the crRNA tracrRNA binding region. The sgRNA may contain, from 5'to 3', an unnatural guide nucleic acid sequence capable of hybridizing to the cell's target sequence, and a tracr sequence. In some cases, the unnatural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. The tracr sequence is a contiguous nucleotide of at least about 60-100 of the native tracrRNA sequence (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). May have at least about 80% with respect to. The tracr sequence is a contiguous nucleotide of SEQ ID NO: 5511 of at least about 60-100 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may have at least about 80% sequence identity with respect to. In some cases, the tracrRNA is at least about 60-90 of SEQ ID NO: 5511 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). 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 relative to consecutive nucleotides. %, At least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA is at least about 60-100 of SEQ ID NO: 5511 (eg, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, or at least about 90). It may be substantially identical to a contiguous nucleotide. The tracrRNA may comprise SEQ ID NO: 5511.

In some cases, at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may contain a sequence having at least about 80% identity to SEQ ID NO: 5475. .. The sgRNA is 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%, relative to SEQ ID NO: 5475. It may contain sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may contain a sequence that is substantially identical to SEQ ID NO: 5475.

In some cases, the system may contain two different sgRNAs that target the first and second regions for cleavage at the target DNA locus, where the second region is the second. It is 3'for the region of 1. In some cases, the system may have at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides from 5'to 3'and 5'in the first region. A first homology arm containing the sequence of, a synthetic DNA sequence of at least about 10 nucleotides, and at least about 20 (eg, at least about 40, 80, 120, 150, 200, 300, 3'in the second region. It may include a single-stranded or double-stranded DNA repair template containing a second homology arm containing a sequence of 500, or 1 kb) nucleotides.

In another aspect, the present disclosure provides a method for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including the enzyme disclosed herein and at least one synthetic guide RNA (sgRNA), to the target nucleic acid locus. But it may be. The enzyme may form a complex with at least one sgRNA and may modify the desired target nucleic acid locus when the complex binds to the desired target nucleic acid locus. Delivering the enzyme to the locus may include transfecting the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include electroporating the cell with the system or nucleic acid encoding the system. Delivering the nuclease to the locus may include incubating the system in a buffer containing the nucleic acid containing the desired locus. In some cases, the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target nucleic acid locus may include genomic DNA, viral DNA, viral RNA, or bacterial DNA. The target nucleic acid locus may be intracellular. The target nucleic acid locus may be in vitro. The target nucleic acid locus may be in eukaryotic cells or in prokaryotic cells. The cells may be animal cells, human cells, bacterial cells, paleobacterial cells, or plant cells. The enzyme may induce single-strand or double-strand breaks at or near the desired target locus.

Where the locus of the target nucleic acid can be intracellular, the enzyme is at least about 75% (eg, at least about 90%, at least about 91%, at least) relative to any one of SEQ ID NOs: 3569-3637. Enzymes with a RuvC_III domain with identity of 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%). It may be supplied as a nucleic acid containing an open reading frame to encode. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to SEQ ID NO: 5587, or at least about 30%, at least about 35%, or at least relative to SEQ ID NO: 5587. 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 It may contain variants having about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. In some cases, the nucleic acid comprises a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be supplied as a capped mRNA containing the open reading frame encoding the endonuclease. The endonuclease may be provided as a translated polypeptide. The at least one engineered sgRNA may be supplied as a deoxyribonucleic acid (DNA) comprising a gene sequence encoding the at least one engineered sgRNA operably linked to the ribonucleic acid (RNA) pol III promoter. .. In some cases, the organism may be a eukaryote. In some cases, the organism may be a fungus. In some cases, the organism may be human.

Example 1. -Metagenomic analysis for novel proteins Metagenomic samples were collected from sediments, soil, and animals. Deoxyribonucleic acid (DNA) was extracted using the Zymobilics DNA mini-prep kit and sequenced on Illumina HiSeqR 2500. Samples were taken with the consent of the owner. Raw sequence data from public sources included sequences of animal microbiomes, sediments, soils, hot springs, hydrothermal vents, oceans, peat swamps, permafrost, and sewage. Metagenomic sequence data were retrieved using a hidden Markov model generated based on known Cas protein sequences, including Type II Cas effector proteins. The novel effector proteins identified by the search were aligned with known proteins to identify potential active sites. This metagenomic workflow elucidates a family of class II, type II CRISPR endonucleases MG1, MG2, MG3, MG4, MG6, MG14, MG15, MG16, MG18, MG21, MG22, and MG23 described herein. did.

Example 2 A. -Discovery of the MG1 family of the CRISPR system When the metagenomic analysis data of Example 1 were analyzed, initially 6 members (MG1-1, MG1-, respectively recorded as SEQ ID NOs: 5, 6, 1, 2, and 3 respectively). 2. A new cluster of previously undescribed putative CRISPR systems including MG1-3, MG1-4, MG1-5, and MG1-6) has been identified. This family features enzymes with HNH and RuvC domains. The RuvC domain of this family has a RuvC_III moiety that is less homologous to previously described Cas9 family members. Although the initial family members have up to 56.8% identity, all six enzymes have the RuvC_III portion of the RuvC domain branched, RHHALDAMV (SEQ ID NO: 5615), KHHALDAMC (SEQ ID NO: 5616). ), Or has a common motif of KHHALDAIC (SEQ ID NO: 5617). These motifs are not found in the other Cas9-like enzymes described. The sequences of proteins and nucleic acids corresponding to these new enzymes and their associated subdomains are presented in the sequence listing. The putative tracrRNA sequence has been identified based on its relative positional relationship with other genes and is presented as SEQ ID NO: 5476-5479. The enzymatic system appears to be derived from Phyllum Verrucomicrobiota, Phylum Candidatus Peregrinibacteria, or Phylum Candidatus Melainabacteria, based on the sequence of 16S rRNA from the phylum bin containing the CRISPR system. The sequence of 16S rRNA is presented as SEQ ID NO: 5592-5596. A detailed domain-level alignment of the CRISPR system sequence that evokes the features described by Shmakov et al. (Mol Cell. 2015 Nov 5; 60 (3): 385-97) is shown in FIGS. 9A, 9B, 9C, 9D, 9E, 9F, It is drawn on 9G and 9H. Comparing MG1-1, 1-2, and 1-3 with additional proprietary protein datasets reveals additional protein sequences with a similar architecture presented as SEQ ID NO: 7-319. rice field. The sequences of these MG1 proteins revealed additional MG1 motifs as set forth in SEQ ID NO: 5618-5632.

Example 2 B. -Discovery of the MG2 family of the CRISPR system Analyzing the data from the metagenomic analysis of Example 1, so far it contains 6 members (MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6). A new cluster of putative CRISPR systems not listed in is revealed. The protein and nucleic acid sequences corresponding to these new enzymes and exemplary subdomains are presented as SEQ ID NOs: 320, 322-325. Estimated tracrRNA sequences have been identified within the operon from their relative positional relationships with other genes and are presented as SEQ ID NOs: 5490, 5492-5494, and 5538. Detailed domain-level alignments of these sequences vs. Cas9 are depicted in FIG. 7 as shown by Shmakov et al. (Mol Cell. 2015 Nov 5; 60 (3): 385-97.).

Comparing the combination of MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6 with an additional proprietary protein dataset, the similarities presented as SEQ ID NOs: 321 and 326-420. Additional protein sequences with the same architecture have been revealed. A common motif among MG2 family members is presented as SEQ ID NO: 5631-5638.

Example 2 C.I. -Discovery of MG3 family of CRISPR system Analysis of the data of the metagenome analysis of Example 1 revealed a newly undescribed estimated CRISPR system: MG3-1. The corresponding amino acid sequences of this new enzyme and its exemplary subdomains are presented as SEQ ID NOs: 424, 2245, and 4059. A putative tracrRNA-containing sequence has been identified based on its proximity to other elements of the operon and is included as SEQ ID NO: 5498. A detailed domain-level alignment of this sequence with Cas9 from Actinomyces naeslundii is shown in FIG.

Comparing MG3-1 with other proprietary protein datasets revealed additional protein sequences with similar structures, presented as SEQ NO: 421-423, 425-431.

Example 2D. -Discovery of MG4, 7, 14, 15, 16, 18, 21, 22, 23 families of CRISPR system When the data of metagenomic analysis of Example 1 is analyzed, 9 families (MG4-5, MG7-2) of 1 member each are analyzed. , MG14-1, MG15-1, MG16-2, MG18-1, MG21-1, MG22-1, MG23-1), revealing a new cluster of previously undescribed estimated CRISPR systems. rice field. The sequences of proteins and nucleic acids corresponding to these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 432, 669, 678, 930, 1093, 1354, 1512, 1656, 1756. Estimated tracr-containing sequences were identified for each family based on their proximity to other elements of the operon. Each of these sequences is presented in the sequence listing as SEQ ID NO: 5503-5511.

Comparing MG4-5, MG7-2, MG14-1, MG15-1, MG16-2, MG18-1, MG21-1, MG22-1, MG23-1 with other proprietary protein datasets, SEQ NO. : Additional with similar architectures presented as 433-660, 670-677, 679-929, 931-1092, 1094-1353, 1355-1511, 1513-1655, 1657-1755, and 1757-1826. The protein sequence was revealed. Common motifs in these sets of CRISPR system nucleases are SEQ ID NO: 5649 for MG4, SEQ ID NO: 5650-5667 for MG14, SEQ ID NO: 5668-5675 for MG15, and SEQ ID NO: 5676- for MG16. 5678, SEQ ID NO: 5679-5686 for MG18, SEQ ID NO: 5678-5693 and SEQ ID NO: 5674-5675 for MG21, SEQ ID NO: 5694-569 for MG22, and SEQ ID NO: 5700-5717 for MG23. It has been presented.

Example 3. -Prophetic --- Determination of Protospacer-Adjacent Motif.
The experiment was carried out by Karvelis et al. Methods. Protospacer flanking motifs for novel enzymes described herein, performed as in any of the examples of 2017 May 15; 121-122: 3-8 (incorporated herein as a whole by reference). PAM) Identify sequence specificity and allow targeting of optimal synthetic sequences.

In one example (in vivo screen), cells having a plasmid encoding any of the enzymes described herein and a guide RNA targeting a protospacer are a plasmid library containing an antibiotic resistance gene, and random. It is co-transformed with a plasmid sequence adjacent to the plasmidized PAM sequence. The plasmid containing the functional PAM is enzymatically cleaved, leading to cell death. Deep sequencing of a pool of enzyme cleavage-resistant plasmids isolated from surviving cells reveals a set of depleted plasmids containing PAM that allows functional cleavage.

In another example (in vitro screening), the PAM library in the form of a DNA plasmid or strand repeat is an RNP complex assembled in vitro or in cytolytic (eg, enzyme, tracrRNA and crRNA, or enzyme and hybrid sgRNA). (Including) exposed to cutting. Free DNA ends resulting from a successful cleavage event are captured by adapter ligation and then exposed to PCR amplification of the product on the PAM side. The library of amplified functional PAMs is subjected to deep sequencing to identify PAMs that license DNA cleavage.

Example 4. -Predictive--Examples of use of the synthetic CRISPR system described herein in mammalian cells for genome editing i) Cell-compatible C-terminal nuclear localization sequences (eg, SV40 for human cells) An ORF encoding a codon-optimizing enzyme under a cell-compatible promoter with NLS) and a suitable polyadenylation signal (eg, TK pA signal for human cells), and (ii) a suitable polymerase III. Under a promoter (eg, U6 promoter in the case of mammalian cells), sgRNA (a 5'sequence starting with G, followed by a complementary targeting nucleic acid sequence 20 nt targeting genomic DNA, followed by Example 3 A DNA / RNA sequence encoding an ORF, which has a 3'tracr binding sequence, a linker, and a tracrRNA sequence) is prepared with the corresponding compatible PAM identified via. In some embodiments, these sequences are prepared on the same or separate plasmid vectors, which are transfected into eukaryotic cells via appropriate techniques. In some embodiments, these sequences are prepared as separate DNA sequences, which are transfected into cells or microinjected. In some embodiments, these sequences are prepared as synthetic RNA or in vitro transcribed RNA that is transfected or microinjected into cells. In some embodiments, these sequences are translated into proteins, transfected into cells, or microinjected.

Regardless of which transfection method is selected, (i) and (ii) are introduced into cells. The incubation period is allowed to elapse so that the enzyme and / or sgRNA is transcribed and / or translated into the active form. After the incubation period, genomic DNA in the vicinity of the targeting sequence is analyzed (eg, by sequencing). As a result of enzyme-mediated cleavage and non-homologous end binding, indels are introduced into genomic DNA near the targeting sequence.

In some embodiments, (i) and (ii) are homologous by being introduced into cells with a third repair nucleotide encoding a region of the genome flanking a cleavage site with a size of 25 bp or greater. Oriented restoration is facilitated. Within these flanking sequences may be a single base pair mutation, a functional gene fragment, an exogenous or natural gene for expression, or multiple genes that make up a biochemical pathway. ..

Example 5. -Predictive--Use of the synthetic CRISPR system described herein In vitro Any of the enzymes described herein is cloned into a suitable E. coli expression plasmid containing a purification tag and recombined with E. coli. It is expressed and purified using a recombinant tag. RNA containing 5'G followed by a 20 nt targeting and PAM sequence, a compatible crRNA tracrRNA binding region, a GAAA linker, and a compatible tracrRNA was synthesized by a suitable solid phase RNA synthesis method. To. The recombinant enzyme and sgRNA were formulated with a suitable cleavage buffer containing Mg2 + (eg, 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl ₂ , 1 mM DTT, 5% glycerol) and with the targeting sequence. The reaction is initiated by introducing a target DNA containing a sequence complementary to the PAM sequence. DNA cleavage is monitored by appropriate assays (eg, agarose gel electrophoresis followed by ethidium bromide staining (or similarly acting DNA inserts) and UV visualization).

Example 6. -(General Protocol) PAM Sequence Identification / Confirmation of Endonucleases Described herein PAM sequences were randomly generated that could be cleaved with putative endonucleases expressed on E. coli lysate-based expression systems (myTXTL, Arbor Biosciences). It was determined by sequencing a plasmid containing the PAM sequence. In this system, E. coli codon-optimized nucleotide sequences were transcribed and translated from PCR fragments under the control of the T7 promoter. A second PCR fragment with a tracr sequence under the T7 promoter and a minimal CRISPR array consisting of the T7 promoter followed by a repeat-spacer-repeat sequence was transcribed in the same reaction. Successful expression of endonucleases and tracr sequences in the TXTL system and subsequent CRISPR array treatment yielded an active CRISPR nuclease complex in vitro.

A library of target plasmids containing a spacer sequence matching the minimal array followed by a mixed base of 8N (estimated PAM sequence) was incubated with the output of the TXTL reaction. After 1-3 hours, the reaction was stopped and DNA was recovered using a DNA cleanup kit such as Zymo DCC, AMPure XP beads, QiaQuick and the like. The adapter sequence was blunt-ended ligated to DNA with the active PAM sequence cleaved by the endonuclease, while the uncleaved DNA was not available for ligation. The DNA segment containing the active PAM sequence was then amplified by PCR using primers specific for the library and adapter sequence. The PCR amplification product was separated on the gel to identify the amplicon corresponding to the cleavage event. The amplified segment of the cleavage reaction was also used as a template for preparing the NGS library. Sequencing the resulting library, which was a subset of the first 8N library, revealed a sequence containing the exact PAM of the active CRISPR complex. In the PAM test with a single RNA construct, the same procedure was repeated except that the RNA transcribed in vitro was added with the plasmid library and the tracr / minimal CRISPR array template was omitted. For the endonucleases for which the NGS library was prepared, the representation of seqLogo (eg, Huber et al. Nat Methods. 2015 Feb; 12 (2): 115-21) was constructed and FIGS. 27, 38, 29, 30, 31 , 32, 33, 34, and 35. The seqLogo module used to construct these representations takes a position weight matrix of DNA sequence motifs (eg, PAM sequences) and plots the corresponding sequence logos introduced by Schneider and Stephens. (See, for example, Schneider et al. Nucleic Acids Res. 1990 Oct 25; 18 (20): 6097-100). The characters representing the sequences in the seqLogo representation are superimposed on each other for each position of the aligned sequence (eg, PAM sequence). The height of each letter is proportional to its frequency, and the letters are rearranged so that the most common one is on top.

Example 7. -(General protocol) RNA folding of tracrRNA and sgRNA structures The folding structure of the guide RNA sequence at 37 ° C. is described in Andronescu et al. Bioinformatics. 2007 Jul 1; 23 (13): Calculated using the method i19-28. Exemplary sgRNA predictive structures described herein are shown in FIGS. 21, 22, 23, 24, 25, and 26.

Example 8. -(General protocol) In vitro cleavage efficiency endonuclease of MG CRISPR complex was expressed in protease-deficient E. coli strain B as a His-tagged fusion protein from the inducible T7 promoter. Cells expressing the His-tagged protein are lysed with ultrasound and the His-tagged protein is subjected to Ni-NTA affinity chromatography on a HisTrap FF column (GE Lifescience) on an AKTA Avant FPLC (GE Lifecare). Purified. The eluate was decomposed by SDS-PAGE on an acrylamide gel (Bio-Rad) and stained with InstantBlue Ultrafast Coomassie (Sigma-Aldrich). Purity was determined using protein band concentration measurements using ImageLab software (Bio-Rad). The purified endonuclease was dialyzed against 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.

The target DNA containing the spacer sequence and the PAM sequence (eg, as determined in Example 6) was constructed by DNA synthesis. One representative PAM was selected for testing when the PAM had a degenerate base. The target DNA contained 2200 bp linear DNA obtained from the plasmid by PCR amplification with PAM located 700 bp from one end and spacers. Successful cleavage yielded 700 bp and 1500 bp fragments. The target DNA, a single RNA transcribed in vitro, and the purified recombinant protein are combined with a cleavage buffer containing excess protein and RNA (10 mM Tris, 100 mM NaCl, 10 mM MgCl ₂ ) from 5 minutes. Incubated for 3 hours, usually 1 hour. The reaction was stopped by the addition of RNAse A and incubation at 60 minutes. The reactants are then degraded on a 1.2% TAE agarose gel and the percentage of target DNA cleaved with ImageLab software is quantified.

Example 9. -(General Protocol) Testing the Genome Cleavage Activity of the MG CRISPR Complex in E. coli E. coli does not have the ability to efficiently repair double-stranded DNA cleavage. Therefore, cleavage of genomic DNA can be a fatal event. Utilizing this phenomenon, endonuclease activity was tested in E. coli by recombinantly expressing endonucleases and tracrRNAs in target strains in which a spacer / target sequence and a PAM sequence were incorporated into genomic DNA.

In this assay, the PAM sequence is specific to the endonuclease under test as determined by the method described in Example 6. The sgRNA sequence was determined based on the expected structure of the traceRNA sequence. Starting from the 5'end of the repeat, a repeat-anti-repeat pair of 8-12 bp (typically 10 bp) was selected. The 3'end of the remaining repeats and the 5'end of tracrRNA were replaced with tetraloop. Generally, the tetraloop was GAAA, but other tetraloops can be used, especially if the GAAA sequence is expected to inhibit folding. In such a case, TTCG Tetraloop was used.

A recombinant strain in which the PAM sequence was incorporated into genomic DNA was transformed with DNA encoding an endonuclease. The transformant is then chemically matched to give 50 ng of a single guide RNA that is specific for the target sequence (“on target”) or non-target (“non target”). Used to transform. After heat shock, the transformants were recovered in SOC at 37 ° C. for 2 hours. The nuclease efficiency was then determined by a 5-fold dilution series cultured in induction medium. Colonies were quantified from the intriplicate dilution series.

Example 10. -(General Protocol) Testing the Genome Cryptonic Activity of the MG CRISPR Complex in Mammalian Cells To show the targeting and cleaving activity in Mammalian cells, the MG Cas effector protein sequence was presented in two mammalian expression vectors: ( A) C-terminal SV40 NLS and 2A-GFP tags were tested, and (b) two SV40 NLS sequences, one at the N-terminus and one at the C-terminus, without the GFP tag. In some examples, the nucleotide sequence encoding the endonuclease is codon-optimized for expression in mammalian cells.

The corresponding single guide RNA sequence (sgRNA) to which the targeting sequence has been added is cloned into a second mammalian expression vector. Two plasmids are cotransfected into HEK293T cells. 72 hours after cotransfection of the expression plasmid and sgRNA targeting plasmid into HEK293T cells, DNA is extracted and used to create the NGS-library. To demonstrate the efficiency of enzyme targeting in mammalian cells, percent NHEJ is measured via the indel in sequencing the target site. At least 10 different target sites were selected to test the activity of each protein.

Example 11. -Characterization of MG1 family members PAM specificity, tracrRNA / sgRNA validation

The target endonuclease activity of the MG1 family endonuclease system was confirmed using the myTXTL system described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in Figure 17-20. Amplification products are MG1-4 (double guide: see gel 1, lane 3, single guide: see gel 6, lane 2), MG1-5 (gel 2, lane 10), MG1-6 (double). Guide: see gel 5, lane 6, single guide: see gel 6, lane 5, MG1-7 (double guide: see gel 3, lane 13, single guide: see gel 3, lane 2) ) (Each protein SEQ ID NO: 1-4) was observed. Sequencing of the PCR products revealed the active PAM sequences of these enzymes, as shown in Table 2.

Synthetic single guide RNAs (sgRNAs) are designed based on the sequence of tracrRNA and the expected structure and are presented as SEQ ID NO: 5461-5464. The PAM sequence screen of Example 6 was repeated with sgRNA. The results of this experiment are also shown in Table 2 and show that the use of sgRNA changed the specificity of PAM slightly.

Targeted endonuclease activity in vitro

The in vitro activity of the MG1-4 endonuclease system (with sgRNA SEQ ID NO: 5461, protein SEQ ID NO: 1) on target DNA with PAM sequence CAGGAAGG was demonstrated using the method of Example 8. The single guide sequence reported above (SEQ ID NO: 5461) was used and the Ns of the sequence was replaced to vary the length of the spacer / targeting sequence in the range of 18-24 nt. The results are shown in FIG. The left panel shows a gel demonstrating DNA cleavage by MG1-4 combined with a corresponding single guide sgRNA with different targeted sequence lengths (18-24 nt), and the right panel quantified the same data as a bar graph. Show things. This data demonstrated that the 18-24 nucleotide targeting sequence was functional in the MG1-4 / sgRNA system.

Targeted endonuclease activity in bacterial cells

The in vivo activity of the MG1-4 endonuclease system (protein SEQ ID NO: 1, sgRNA SEQ ID NO: 5461) was tested using the PAM sequence CAGGAAGG as in Example 9. Transformed E. coli are plated with serial dilutions and the results (the left panel shows the serial dilutions of E. coli and the right panel shows the quantified growth) are presented in FIG. Significant reduction in proliferation of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by endonucleases within E. coli cells.

Targeted endonuclease activity in mammalian cells

The method of Example 10 was used to demonstrate targeting and cleavage activity in mammalian cells. Open reading frames encoding the sequences of MG1-4 (protein SEQ ID NO: 5527) and MG1-6 (protein SEQ ID NO: 5259) are those having a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB). , GFP-tagged and cloned into two mammalian expression vectors, one at the N-terminus and one at the C-terminus (E. coli pMG5-BB). For MG1-6, the open reading frame was further codon-optimized for mammalian expression (SEQ ID NO: 5589) and cloned into a 2-NLS plasmid backbone (MG-16hs). The results of this experiment are shown in FIG. The endonuclease expression vector is HEK293T with a second vector for expressing an sgRNA (eg, SEQ ID NO: 5512 or 5515) having an endonuclease-specific tracr sequence and a guide sequence selected from Tables 3-4. The cells were cotransfected. Seventy-two hours after cotransfection, DNA was extracted and used to prepare the NGS-library. Cleavage activity was detected by the appearance of an internal deletion (NHEJ remnant) in close proximity to the sequence at the target site. In order to demonstrate the targeting efficiency of the enzyme in mammalian cells, the proportion of NHEJ was measured via the indel in the sequence of the target site and is shown in FIG.

Example 12. -Characterization of MG2 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG2 family members was confirmed on the myTXTL system as described in Example 6. The results of this assay are shown in FIGS. 17-20. In the assay shown in FIGS. 17-20, the active protein that successfully cleaves the library produces a band of approximately 170 bp in the gel. Amplification products were observed for MG2-1 (see Gel 2, Lane 11 and Gel 4, Lane 6) and MG2-7 (see Gel 11, Lane 10) (SEQ ID NOS: 320 and 321 respectively). Sequencing of the PCR product revealed the active PAM sequences shown in Table 5 below.

Targeted endonuclease activity in bacterial cells

The in vivo activity of the MG2-7 endonuclease system with sgRNA (endonuclease SEQ ID NO: 321; sgRNA SEQ ID NO: 5465) and the AGCGTAAG PAM sequence was confirmed using the method described in Example 9. Transformed E. coli are plated with serial dilutions and the results (the left panel shows the serial dilutions of E. coli and the right panel shows the quantified growth) are presented in FIG. Significant reduction in proliferation of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved in E. coli cells by MG1-4 endonuclease. There is.

Example 13. -Characteristics of MG3 family members

Verification of PAM specificity, tracrRNA / sgRNA

Targeted endonuclease activity of MG3 family members was confirmed using the tracr sequence and the CRISPR array using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. MG3-6 (double guide: see gel 2, lane 8, single guide: see gel 3, lane 3), MG3-7 (double guide: see gel 2, lane 3, single guide: gel) 3. Amplification products were observed in MG3-8 (dual guides: see gel 9, lane 5) (see lanes 4) (SEQ ID NOs: 421, 422, and 423, respectively). Sequencing of the PCR product revealed the active PAM sequences shown in Table 6 below.

Synthetic single guide RNAs (sgRNAs) are designed based on the sequence of tracrRNA and the expected structure and are presented as SEQ ID NO: 5466-5467. The PAM sequence screen of Example 6 was repeated with sgRNA. The results of this experiment are also shown in Table 6 and show that the use of sgRNA changed the specificity of PAM slightly.

Targeted endonuclease activity in vitro

In vitro activity of MG3-6 (endonuclease SEQ ID NO: 421) was demonstrated with the PAM sequence GTGGGTTA using the method of Example 8. The single guide sequence reported above (SEQ ID NO: 5466) was used, substituting the Ns of the sequence and varying the length of the spacer / targeting sequence in the range of 18-24 nt. The result is shown in FIG. The upper panel shows a gel demonstrating DNA cleavage by MG3-6 in combination with various sgRNAs with different targeted sequence lengths (18-24 nt), and the lower panel shows the same data quantified as a bar graph. .. This data demonstrated that the 18-24 nucleotide targeting sequence was functional in the MG3-6 / sgRNA system.

Targeted endonuclease activity in bacterial cells

The in vivo activity of the MG3-7 endonuclease system (protein SEQ ID NO: 422, sgRNA SEQ ID NO: 5467) was tested using the PAM sequence TGGACCTG using the method of Example 9. Transformed E. coli are plated with serial dilutions and the results (upper panel showing serial dilutions of E. coli and lower panel showing quantified growth) are presented in FIG. Significant reduction in proliferation of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by the MG3-7 endonuclease system.

Targeted endonuclease activity in mammalian cells

The method of Example 10 was used to demonstrate targeting and cleavage activity in mammalian cells. The open reading frames encoding the sequence of MG3-7 (protein SEQ ID NO: 422) are those with C-terminal SV40 NLS and 2A-GFP tag (E. coli MG-BB) and those without GFP tag and 1 at the N-terminus. It was cloned into two mammalian expression vectors, one with one and two NLS at the C-terminus (E. coli pMG5-BB). The endonuclease expression vector was co-transfected into HEK293T cells with a second vector for expressing the sgRNA described above using the guide sequence selected from Table 7. The results of this experiment are shown in FIG. Seventy-two hours after cotransfection, DNA was extracted and used to prepare the NGS-library. Cleavage activity was detected by the appearance of an internal deletion (NHEJ remnant) in the vicinity of the target site. The results are shown in FIG.

The target sites encoded on the sgRNA plasmid are shown in Table 7 below.

Example 13. -Characterization of MG4 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of the MG4 family endonuclease system was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG4-2 (see Double Guide: Gel 2, Lane 9, Single Guide: Gel 10, Lane 7) (SEQ ID NO: 432). Sequencing of the PCR product revealed the active PAM sequences shown in Table 8 below.

Example 14. Characterization of MG14 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG14 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG14-1 (see Double Guide: Gel 2, 1 Lane 4, Single Guide: Gel 3, Lane 8) (SEQ ID NO: 678). Sequencing of the PCR product revealed the active PAM sequence specificity shown in Table 9 below.

Targeted endonuclease activity in bacterial cells

The in vivo activity of the MG14-1 endonuclease system with sgRNA (endonuclease SEQ ID NO: 678; sgRNA SEQ ID NO: 5469) and the GGCGGGGA PAM sequence was confirmed using the method described in Example 9. Transformed E. coli are plated with serial dilutions and the results (the left panel shows the serial dilutions of E. coli and the right panel shows the quantified growth) are presented in FIG. Significant reduction in proliferation of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved in E. coli cells by MG1-4 endonuclease. There is.

Example 15. -Characterization of MG15 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG15 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG15-1 (see Double Guide: Gel 2, Lane 7, Single Guide: Gel 3, Lane 9) (SEQ ID NO: 930). Sequencing of the PCR product revealed the active PAM sequence specificity detailed in Table 10 below.

In vitro activity

The in vitro activity of the MG15-1 endonuclease system (protein SEQ ID NO: 930, sgRNA SEQ ID NO: 5470) was tested using the PAM sequence GGGTCAAA using the method of Example 8. The single guide sequence reported above (SEQ ID NO: 5470) was used and the Ns of the sequence was replaced to vary the length of the spacer / targeting sequence in the range of 18-24 nt. The result is shown in FIG. The upper panel shows a gel demonstrating DNA cleavage by MG15-1 in combination with various sgRNAs with different targeted sequence lengths (18-24 nt), and the lower panel shows the same data quantified as a bar graph. .. This data demonstrated that the 18-24 nucleotide targeting sequence was functional in the MG15-1 / sgRNA system.

Targeted endonuclease activity in bacterial cells

The in vivo activity of the MG15-1 endonuclease system with sgRNA (endonuclease SEQ ID NO: 930; sgRNA SEQ ID NO: 5470) and the GGGTCAAA PAM sequence was confirmed using the method described in Example 9. Transformed E. coli are plated with serial dilutions and the results (the left panel shows the serial dilutions of E. coli and the right panel shows the quantified growth) are presented in FIG. Significant reduction in proliferation of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved in E. coli cells by MG1-4 endonuclease. There is.

Example 16. -Characterization of MG16 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG16 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG16-2 (see Gel 11, Lane 17) (SEQ ID NO: 1093). Sequencing of the PCR product revealed the active PAM sequence specificity detailed in Table 11 below.

Example 17. -Characterization of MG18 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG18 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG18-1 (see Double Guide: Gel 2, Lane 9, Single Guide: Gel 11, Lane 12) (SEQ ID NO: 1354). Sequencing of the PCR product revealed the active PAM sequence specificity detailed in Table 12 below.

Example 18. -Characterization of MG21 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of the MG21 family was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG21-1 (see Gel 11, Lane 2) (SEQ ID NO: 1512). Sequencing of the PCR product revealed the active PAM sequence specificity detailed in Table 13 below.

Example 19. -Characterization of MG22 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG22 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. In the assay shown in FIGS. 17-20, the active protein that successfully cleaves the library produces a band of approximately 170 bp in the gel. Amplification products were observed for MG22-1 (see Gel 11, Lane 3) (protein SEQ ID NO: 1656). Sequencing of the PCR product revealed the active PAM sequence specificity detailed in Table 14 below.

Example 20. -Characterization of MG23 family members PAM specificity, validation of tracrRNA / sgRNA

Targeted endonuclease activity of MG23 family members was confirmed using the myTXTL system as described in Example 6. In this assay, PCR amplification of the cleaved target plasmid gives a product that migrates at about 170 bp in the gel, as shown in FIGS. 17-20. Amplification products were observed for MG23-1 (see Gel 11, Lane 4) (SEQ ID NO: 1756). Sequencing of PCR products revealed active PAM sequence specificity for these enzymes, detailed in Table 15 below.

本開示のシステムは、例えば、核酸編集（例えば、遺伝子編集）、核酸分子への結合（例えば、配列特異的結合）など、様々な用途に使用することができる。このようなシステムは、例えば、ウイルスゲノムを標的とすることでウイルスを不活性化したり、宿主細胞に感染できないようにしたりするために、価値の高い低分子、高分子、または二次代謝産物を生成するように生物を操作するべく遺伝子を追加したり、代謝経路を変更したりするために、進化的選択のための遺伝子駆動要素を確立するために、バイオセンサーとして外来の低分子およびヌクレオチドによる細胞摂動を検出するために、特定のヌクレオチド配列（例えば、細菌における抗生物質耐性をコードする配列）を標的とするとともに検出するためにプローブと組み合わせた不活性化酵素のように、疾患を引き起こす遺伝的要素を検出するための診断ツールとして（例えば、逆転写されたウイルスＲＮＡまたは疾患を引き起こす突然変異をコードする増幅されたＤＮＡ配列の切断を介して）、被験体において疾患を引き起こす可能性のある遺伝的に受け継がれた突然変異をアドレス指定（例えば、除去または置換）して、遺伝子を不活性化することで細胞内での遺伝子の機能を確認するために使用されてもよい。

The systems of the present disclosure can be used for a variety of applications, such as nucleic acid editing (eg, gene editing), binding to nucleic acid molecules (eg, sequence-specific binding), and the like. Such systems provide high-value small, high-molecular-weight, or secondary metabolites, for example, to inactivate the virus by targeting the viral genome or prevent it from infecting host cells. By foreign small molecules and nucleotides as biosensors to establish gene-driving elements for evolutionary selection, to add genes to manipulate organisms to produce, or to alter metabolic pathways. Disease-causing inheritance, such as inactivating enzymes combined with probes to target and detect specific nucleotide sequences (eg, sequences encoding antibiotic resistance in bacteria) to detect cell perturbations. As a diagnostic tool for detecting target elements (eg, through cleavage of reverse transcribed viral RNA or amplified DNA sequences encoding disease-causing mutations), it can cause disease in the subject. It may be used to identify the function of a gene in a cell by addressing (eg, removing or replacing) a genetically inherited mutation to inactivate the gene.

Although preferred embodiments of the 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. The present invention is not intended to be limited by the particular examples provided herein. Although the present invention has been described with respect to the specification described above, the description and illustration of embodiments herein are not intended to be construed in a limited sense. Those skilled in the art will be able to come up with many changes, changes, and substitutions without departing from the invention. Further, it will be appreciated that all aspects of the invention are not limited to the particular depictions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the invention described herein may be utilized in the practice of the invention. Therefore, the invention is intended to extend to any such alternatives, modifications, variants, or equivalents. The following claims define the scope of the invention, and it is intended that the methods and structures within the scope of this claim and its equivalents are embraced therein.

Claims (170)

An engineered nuclease system, said engineered nuclease system.
(A) An endonuclease containing a RuvC_III domain and an HNH domain, wherein the endonuclease is derived from a refractory microorganism, where the endonuclease is a class 2 type II Cas endonuclease. , Endonucleases,
(B) An engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, wherein the engineered guide ribonucleic acid structure is:
(I) A guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence and
(Ii) Containing an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease.
Manipulated nuclease system. 13. The RuvC_III domain according to claim 1, wherein the RuvC_III domain comprises a sequence having at least 70%, at least 75%, at least 80%, or at least 90% sequence identity to any one of SEQ ID NOs: 1827-3637. Manipulated nuclease system. An engineered nuclease system, said engineered nuclease system.
(A) An endonuclease containing a RuvC_III domain having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637.
(B) An engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, wherein the engineered guide ribonucleic acid structure is:
(I) A guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence and
(Ii) Containing an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease.
Manipulated nuclease system. An engineered nuclease system, said engineered nuclease system.
(A) An endonuclease configured to bind to a protospacer flanking motif (PAM) sequence comprising SEQ ID NO: 5512-5537, wherein the endonuclease is a class 2 type II Cas endonuclease. , Endonucleases,
(B) An engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, wherein the engineered guide ribonucleic acid structure is:
(I) A guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence and
(Ii) Containing an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease.
Manipulated nuclease system. The engineered nuclease system of claim 4, wherein the endonuclease is derived from a refractory microorganism. The engineered nuclease system of any one of claims 4-5, wherein the endonuclease has not been engineered to bind to a different PAM sequence. The endonucleases are Cas9 endonucleases, Cas14 endonucleases, Cas12a endonucleases, Cas12b endonucleases, Cas12c endonucleases, Cas12d endonucleases, Cas12e endonucleases, Cas13a endonucleases, Cas13b endonucleases, Cas13c endonucleases, or Cas13d endonucleases. No, the engineered nuclease system of claim 4. The engineered nuclease system of claim 4, wherein the endonuclease has less than 80% identity to Cas9 endonuclease. The engineered nuclease system of any one of claims 3-8, wherein the endonuclease further comprises an HNH domain. The tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to about 60-90 contiguous nucleotides selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. , The engineered nuclease system according to any one of claims 1-9. An engineered nuclease system, said engineered nuclease system.
(A) An manipulated guide ribonucleic acid structure, wherein the manipulated guide ribonucleic acid structure is:
(I) A guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence and
(Ii) A tracr ribonucleic acid sequence configured to bind to an endonuclease, wherein the tracr ribonucleic acid sequence is selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. Includes a tracr ribonucleic acid sequence, which comprises a sequence having at least 80% sequence identity to about 60-90 contiguous nucleotides.
Manipulated guide ribonucleic acid structure and
(B) Containing a Class 2 Type II Cas endonuclease configured to bind to the engineered guide ribonucleic acid.
Manipulated nuclease system. The manipulation according to any one of claims 1-3 or 11, wherein the endonuclease is configured to bind to a protospacer flanking motif (PAM) sequence selected from the group comprising SEQ ID NO: 5512-5537. Nuclease system. The engineered nuclease system of any one of claims 1-11, wherein the engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides. The engineered nuclease system of any one of claims 1-11, wherein the engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence. The operation according to any one of claims 1-14, wherein the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. Nuclease system. The engineered nuclease system according to any one of claims 1-15, wherein the guide ribonucleic acid sequence is 15 to 24 nucleotides in length. The engineered according to any one of claims 1-16, wherein the endonuclease comprises one or more nuclear localization sequences (NLS) located proximal to the N-terminus or C-terminus of the endonuclease. Nuclease system. The engineered nuclease system of any one of claims 1-17, wherein the NLS comprises a sequence selected from SEQ ID NO: 5597-5612. From 5'to 3', a first homology arm containing a sequence of at least 20 nucleotides in 5'of the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and 3'of the target deoxyribonucleic acid sequence. The engineered according to any one of claims 1-18, further comprising a single-stranded or double-stranded DNA repair template comprising a second homology arm comprising a sequence of at least 20 nucleotides. Nuclease system. 19. The operation of claim 19, wherein the first homology arm or the second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. Nuclease system. The engineered nuclease system according to any one of claims 1-20, wherein the engineered nuclease system further comprises a source of Mg ²⁺ . The engineered nuclease system according to any one of claims 1-21, wherein the endonuclease and the tracr ribonucleic acid sequence are derived from distinct bacterial species within the same phylum. The engineered nuclease system according to any one of claims 1-22, wherein the endonuclease is derived from a bacterium belonging to the genus Dermabacter. The engineered nuclease system according to any one of claims 1-22, wherein the endonuclease is derived from a bacterium belonging to the phylum Verrucomicrobiota, the phylum Candidatus Peregrinibacteria, or the phylum Candidatus Melainabacteria. The endonuclease is described in any one of claims 1-22, wherein the endonuclease is derived from a bacterium comprising a 16S rRNA gene having at least 90% identity to any one of SEQ ID NOs: 5592-5595. Manipulated nuclease system. The manipulation of any one of claims 1-25, wherein the HNH domain comprises a sequence having at least 70% or at least 80% identity to any one of SEQ ID NOs: 5638-5460. Nuclease system. The engineered nuclease system of any one of claims 1-26, wherein the endonuclease comprises SEQ ID NO: 1-1826 or a variant thereof having at least 55% identity to it. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1827-1830 or SEQ ID NO: 1827-2140. 27 The engineered nuclease system according to any one of 27. The endonuclease comprises a sequence that is at least 70%, 80%, 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3638-3461 or SEQ ID NO: 3638-3954, claim 1-28. The engineered nuclease system according to any one of the above. Any of claims 1-29, wherein the endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5615-5632. The engineered nuclease system according to one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1-4 or SEQ ID NO: 1-319. 30 The engineered nuclease system according to any one of 30. The guide RNA structure is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5461-5464, SEQ ID NO: 5476-5479, or SEQ ID NO: 5476-5489. The engineered nuclease system according to any one of claims 1-31, comprising a sequence. The guide RNA structure comprises an RNA sequence that is expected to contain a hairpin consisting of a stem and a loop, wherein the stem is at least 10, at least 12, or at least 14 base pair ribonucleotides, and the loop. The engineered nuclease system of any one of claims 1-32, comprising an asymmetric bulge within four base pairs. The endonuclease is described in any one of claims 1-33, wherein the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5512-5515 or SEQ ID NO: 5527-5530. Manipulated nuclease system. a) The endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1827.
b) The guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5461 or SEQ ID NO: 5476, and.
c) The engineered nuclease system of any one of claims 1-34, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5512 or SEQ ID NO: 5527. a) The endonuclease contains a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1828.
b) The guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5462 or SEQ ID NO: 5477, and.
c) The engineered nuclease system of any one of claims 1-34, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5513 or SEQ ID NO: 5528. a) The endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1829.
b) The guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5436 or SEQ ID NO: 5478, and.
c) The engineered nuclease system of any one of claims 1-34, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5514 or SEQ ID NO: 5259. a) The endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1830.
b) The guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5464 or SEQ ID NO: 5479, and.
c) The engineered nuclease system of any one of claims 1-34, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5515 or SEQ ID NO: 5530. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2141-2142 or SEQ ID NO: 2141-2241. 27 The engineered nuclease system according to any one of 27. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3955-3965 or SEQ ID NO: 3955-4055. The engineered nuclease system according to any one of 27 or 39. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5632-5638, claim 1-27 or 39. The engineered nuclease system according to any one of -40. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 320-321 or SEQ ID NO: 320-420, claim 1-. 27 or 39-41 according to any one of the engineered nuclease systems. The guide RNA structure is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5465, SEQ ID NO: 5490-5491, or SEQ ID NO: 5490-5494. The engineered nuclease system according to any one of claims 1-27 or 39-42. The guide RNA structure comprises any one of claims 1-27 or 39-43 comprising a tracr ribonucleic acid sequence comprising a hairpin comprising at least 8, at least 10 or at least 12 base pair ribonucleotides. Manipulated nuclease system. 13. Manipulated nuclease system. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2141.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5490, and
c) The engineered nuclease system of any one of claims 1-27 or 39-45, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5531. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2142.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5465 or SEQ ID NO: 5491, and
c) The engineered nuclease system of any one of claims 1-27 or 39-45, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5516. The endonuclease comprises any one of claims 1-27 comprising a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2245-2246. The engineered nuclease system described. One of claims 1-27 or 48, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4059-4060. One of the engineered nuclease systems described. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5639-5648, claim 1-27 or 48. The engineered nuclease system according to any one of -49. Either of claims 1-27 or 48-50, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 424-425. One of the engineered nuclease systems described in The guide RNA structure comprises a sequence that is at least 70%, 80%, 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5498-5499 and SEQ ID NO: 5539, claim 1-27 or. The engineered nuclease system according to any one of 48-51. The guide RNA structure comprises a guide ribonucleic acid sequence that is expected to contain a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. , The tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin at 5'to 3'where the first hairpin has a longer stem than the second hairpin, claimed. The engineered nuclease system according to any one of Items 1-27 or 48-52. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2242-2244 or SEQ ID NO: 2247-2249. 27 The engineered nuclease system according to any one of 27. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4056-4058 and SEQ ID NO: 4061-4063. The engineered nuclease system according to any one of 27 or 54. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5639-5648, claim 1-27 or 54. The engineered nuclease system according to any one of -55. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 421-423 or SEQ ID NO: 426-428. The engineered nuclease system according to any one of 27 or 54-56. The guide RNA structure is at least 70%, 80%, relative to a sequence selected from the group consisting of SEQ ID NO: 5466-5467, SEQ ID NO: 5495-5497, SEQ ID NO: 5500-5502, and SEQ ID NO: 5539. Or the engineered nuclease system of any one of claims 1-27 or 54-57, comprising sequences that are 90% identical. The guide RNA structure comprises a guide ribonucleic acid sequence that is expected to contain a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. , The tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin at 5'to 3'where the first hairpin has a longer stem than the second hairpin, claimed. The engineered nuclease system according to any one of Items 1-27 or 54-58. Either of claims 1-27 or 54-59, wherein the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5517-5518 or SEQ ID NO: 5532-5534. One of the engineered nuclease systems described. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2247.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5500, and
c) The engineered nuclease system of any one of claims 1-27 or 54-60, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5517 or SEQ ID NO: 5532. .. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2248.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5501, and
c) The engineered nuclease system of any one of claims 1-27 or 54-60, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5518 or SEQ ID NO: 5533. .. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2249.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5502, and
c) The engineered nuclease system of any one of claims 1-27 or 54-60, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5534. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2253 or SEQ ID NO: 2253-2481. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4067 or SEQ ID NO: 4067-4295, claim 1-27 or. The engineered nuclease system according to any one of 64. The engineered nuclease system of any one of claims 1-27 or 64-65, wherein the endonuclease comprises a peptide motif of SEQ ID NO: 5649. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 432 or SEQ ID NO: 432-660, claim 1-27 or. The engineered nuclease system according to any one of 64-66. The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5468 or SEQ ID NO: 5503, claim 1-27 or 64. The engineered nuclease system according to any one of -67. The engineered nuclease according to any one of claims 1-27 or 64-68, wherein the endonuclease is configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5519. system. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2253.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5468 or SEQ ID NO: 5503, and
c) The engineered nuclease system of any one of claims 1-27 or 64-69, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5519. The endonuclease comprises any one of claims 1-27 comprising a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2482-2489. The engineered nuclease system described. One of claims 1-27 or 71, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4296-4303. One of the engineered nuclease systems described. Either of claims 1-27 or 71-72, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 661-668. The engineered nuclease system according to one. The endonuclease comprises any one of claims 1-27 comprising a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2490-2498. The engineered nuclease system described. One of claims 1-27 or 74, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4304-4321. One of the engineered nuclease systems described. Either of claims 1-27 or 74-75, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 669-677. The engineered nuclease system according to one. One of claims 1-27 or 74-76, wherein the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5504. One of the engineered nuclease systems described. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2499 or SEQ ID NO: 2499-2750, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4313 or SEQ ID NO: 4313-4564, claim 1-27 or. The engineered nuclease system according to any one of 78. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5650-5667, claim 1-27 or 78. The engineered nuclease system according to any one of -79. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 678 or SEQ ID NO: 678-929, claim 1-27 or. The engineered nuclease system according to any one of 78-80. One of claims 1-27 or 78-81, wherein the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505. The engineered nuclease system described. The engineered nuclease system of any one of claims 1-27 or 78-82, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2499.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5469 or SEQ ID NO: 5505, and
c) The engineered nuclease system of any one of claims 1-27 or 78-83, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535. .. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2751 or SEQ ID NO: 2751-2913, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4565 or SEQ ID NO: 4565-4727, claim 1-27 or. The engineered nuclease system according to any one of 85. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5668-5678, claim 1-27 or 85. The engineered nuclease system according to any one of -86. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 930 or SEQ ID NO: 930-1092, claim 1-27 or. The engineered nuclease system according to any one of 85-87. One of claims 1-27 or 85-88, wherein the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5470 or SEQ ID NO: 5506. The engineered nuclease system described. The endonuclease is described in any one of claims 1-27 or 85-89 configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5521 or SEQ ID NO: 5536. Manipulated nuclease system. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2751.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5470 or SEQ ID NO: 5506, and
c) The engineered nuclease system of any one of claims 1-27 or 85-90, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5521 or SEQ ID NO: 5536. .. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2914 or SEQ ID NO: 2914-3174, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4728 or SEQ ID NO: 4728-4988, claim 1-27 or. 92. The engineered nuclease system according to any one of 92. The endonuclease comprises any one of claims 1-27 or 92-93 comprising at least one, at least two, or at least three peptide motifs selected from the group consisting of SEQ ID NO: 5676-5678. The engineered nuclease system described. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1093 or SEQ ID NO: 1093-1353, claim 1-27 or. The engineered nuclease system according to any one of 92-94. The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5471, SEQ ID NO: 5507, and SEQ ID NO: 5540-5542. , The engineered nuclease system according to any one of claims 1-27 or 92-95. 13. Manipulated nuclease system. The engineered nuclease system of any one of claims 1-27 or 92-97, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5522. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2914.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5471 or SEQ ID NO: 5507, and
c) The engineered nuclease system of any one of claims 1-27 or 92-98, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5522. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3175 or SEQ ID NO: 3175-3330, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 4989 or SEQ ID NO: 4989-5146, claim 1-27 or. The engineered nuclease system according to any one of 100. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5679-5686, claim 1-27 or 100. The engineered nuclease system according to any one of -101. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1354 or SEQ ID NO: 1354-1511, claim 1-27 or. The engineered nuclease system according to any one of 100-102. The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5472 or SEQ ID NO: 5508, claim 1-27 or 100. -The engineered nuclease system according to any one of 103. The endonuclease is described in any one of claims 1-27 or 100-104, configured to bind to a PAM comprising a sequence selected from the group consisting of SEQ ID NO: 5523 or SEQ ID NO: 5537. Manipulated nuclease system. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3175.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5472 or SEQ ID NO: 5508, and
c) The engineered nuclease system of any one of claims 1-27 or 100-105, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5523 or SEQ ID NO: 5537. .. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3331 or SEQ ID NO: 3331-3474, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5147 or SEQ ID NO: 5147-5290, claim 1-27 or. The engineered nuclease system according to any one of 107. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5674-5675 and SEQ ID NO: 5687-5693. The engineered nuclease system according to any one of claims 1-27 or 107-108. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1512 or SEQ ID NO: 1512-1655, claim 1-27 or. The engineered nuclease system according to any one of 107-109. The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5473 or SEQ ID NO: 5509, claim 1-27 or 107. The engineered nuclease system according to any one of -110. The engineered nuclease system of any one of claims 1-27 or 107-111, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5524. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3331.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5473 or SEQ ID NO: 5509, and
c) The engineered nuclease system of any one of claims 1-27 or 107-112, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5524. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3475 or SEQ ID NO: 3475-3568, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5291 or SEQ ID NO: 5291-5389, claim 1-27 or. The engineered nuclease system according to any one of 114. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5694-569, claim 1-27 or 114. The engineered nuclease system according to any one of -115. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1656 or SEQ ID NO: 1656-1755, claim 1-27 or. The engineered nuclease system according to any one of 114-116. One of claims 1-27 or 114-117, wherein the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5474 or SEQ ID NO: 5510. The engineered nuclease system described. The engineered nuclease system of any one of claims 1-27 or 114-118, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5525. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3475.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5474 or SEQ ID NO: 5510, and
c) The engineered nuclease system of any one of claims 1-27 or 114-119, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5525. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3569 or SEQ ID NO: 3569-3637, claim 1-27. The engineered nuclease system according to any one. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 5390 or SEQ ID NO: 5390-5460, claim 1-27 or. The engineered nuclease system according to any one of 121. The endonuclease comprises at least one, at least two, at least three, at least four, or at least five peptide motifs selected from the group consisting of SEQ ID NO: 5700-5717, claim 1-27 or 121-. The engineered nuclease system according to any one of 122. The endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 1756 or SEQ ID NO: 1756-1826, claim 1-27 or. The engineered nuclease system according to any one of 121-123. One of claims 1-27 or 121-124, wherein the guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5475 or SEQ ID NO: 5511. The engineered nuclease system described. The engineered nuclease system of any one of claims 1-27 or 121-125, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5526. a) The endonuclease contains a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3569.
b) The guide RNA structure comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5475 or SEQ ID NO: 5511, and
c) The engineered nuclease system of any one of claims 1-27 or 121-126, wherein the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5526. The engineered nuclease system of any one of claims 1-127, wherein the sequence identity is determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, or the Smith-Waterman homology search algorithm. The sequence identity is conditional using the BLASTUM62 scoring matrix, which sets the gap cost at 3 wordlength (W), 10 extraction (E) parameters, and 11 exhibitions, 1 extension. The engineered nuclease system of claim 128, as determined by the BLASTP homology search algorithm using composition score matrix adjustments. The engineered guide ribonucleic acid polynucleotide, wherein the engineered guide ribonucleic acid polynucleotide is.
a) A DNA-targeted segment that contains a nucleotide sequence that is complementary to the target sequence in the target DNA molecule.
b) A protein-binding segment containing two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex.
Here, the two complementary stretches of said nucleotides are covalently attached to each other at the intervening nucleotides, and
Here, the engineered guide ribonucleic acid polynucleotide forms a complex with an endonuclease containing a RuvC_III domain having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637, said. Configured to target the complex to the target sequence of the target DNA molecule,
Manipulated guide ribonucleic acid polynucleotide. The engineered guide ribonucleic acid polynucleotide according to claim 130, wherein the DNA targeting segment is located at 5'both of the two complementary stretches of the nucleotide. a) The protein binding segment is a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5476-5479 or SEQ ID NO: 5476-5489. Including
b) The protein binding segment is at least 70%, at least 80%, or at least 90% of the sequence selected from the group consisting of (SEQ ID NO: 5490-5491 or SEQ ID NO: 5490-5494) and SEQ ID NO: 5538. Containing sequences with the same identity of
c) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5498-5499.
d) The protein binding segment is a sequence having at least 70%, at least 80%, or at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5495-5497 and SEQ ID NO: 5500-5502. Including
e) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5503.
f) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5504.
g) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5505.
h) Protein binding segments include sequences having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5506.
i) Protein binding segments include sequences having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5507.
j) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5508.
k) The protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5509.
l) The protein binding segment comprises, or contains, a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5510.
m) The engineered according to any of claims 130-131, wherein the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5511. Guide ribonucleic acid polynucleotide. a) The guide ribonucleic acid polynucleotide comprises an RNA sequence comprising a hairpin comprising a stem and a loop, wherein the stem is at least 10, at least 12, or at least 14 base pair ribonucleotides, and the loop. Contains asymmetric bulges within 4 base pairs
b) The guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence that is expected to contain a hairpin containing at least 8, at least 10, or at least 12 base pair ribonucleotides.
c) The guide ribonucleic acid polynucleotide is expected to contain a hairpin having an uninterrupted base pair region containing at least 8 nucleotides of the guide ribonucleic acid sequence and at least 8 nucleotides of the tracr ribonucleic acid sequence. Containing, where the tracr ribonucleic acid sequence comprises a first hairpin and a second hairpin at 5'to 3'where the first hairpin has a stem longer than the second hairpin. Or have
d) The engineered according to any of claims 130-132, wherein the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence that is expected to contain at least two hairpins comprising less than five base pairs of ribonucleotides. Guided ribonucleic acid polynucleotide. A deoxyribonucleic acid polynucleotide encoding the engineered guide ribonucleic acid polynucleotide according to any one of claims 130-133. A nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein said nucleic acid encodes a class 2 type II Cas endonuclease containing a RuvC_III domain and an HNH domain. Here, the class 2 Cas endonuclease is a nucleic acid derived from a refractory microorganism. A nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein said nucleic acid is at least 70% sequence identical to any one of SEQ ID NOs: 1827-3637. A nucleic acid encoding an endonuclease containing a RuvC_III domain having sex. 13. Nucleic acid. The nucleic acid according to any one of claims 135-137, wherein the endonuclease comprises SEQ ID NO: 5571-5591 or a variant thereof having at least 70% sequence identity to them. 13. One of claims 135-138, wherein the endonuclease comprises a sequence encoding one or more nuclear localization sequences (NLS) located proximal to the N-terminus or C-terminus of the endonuclease. Nucleic acid. The nucleic acid of claim 139, wherein the NLS comprises a sequence selected from SEQ ID NO: 5597-5612. The nucleic acid according to any one of claims 135-140, wherein the organism is a prokaryote, a bacterium, a eukaryote, a fungus, a plant, a mammal, a rodent, or a human. The organism is E. coli, where
a) The nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5571-5575.
b) The nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5576-5571.
c) The nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NO: 5578-5580.
d) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5581.
e) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5582.
f) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5583.
g) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5584.
h) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5585.
i) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5586, or
j) The nucleic acid of claim 141, wherein the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5587. The organism is human, where
a) The nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5588 or SEQ ID NO: 5589, or
b) The nucleic acid of claim 141, wherein the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5590 or SEQ ID NO: 5591. A vector containing a nucleic acid sequence encoding a class 2 type II Cas endonuclease containing a RuvC_III domain and an HNH domain, wherein the class 2 type II Cas endonuclease is a vector derived from a refractory microorganism. A vector comprising the nucleic acid according to any one of claims 135-143. The engineered guide ribonucleic acid structure further comprises a nucleic acid encoding an engineered guide ribonucleic acid structure configured to form a complex with the class 2 type II Cas endonuclease.
a) A guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence and
b) A tracr ribonucleic acid sequence configured to bind to the class 2 Cas endonuclease.
The vector according to any one of claims 144-145. The vector according to any one of claims 144-146, wherein the vector is a plasmid, minicircle, CELiD, adeno-associated virus (AAV) -derived virion, or lentivirus. A cell comprising the vector according to any one of claims 144-147. A method for producing an endonuclease, wherein the method comprises the step of culturing the cells of claim 146. A method for binding, cleaving, labeling, or modifying a double-stranded deoxyribonucleic acid polynucleotide, said method.
(A) Class 2 type II Cas endonucleases and class 2 type II Cas endonucleases that form a complex with an engineered guide ribonucleic acid structure configured to bind to said double-stranded deoxyribonucleic acid polynucleotide. Including the step of contacting the double-stranded deoxyribonucleic acid polynucleotide.
(B) The double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer flanking motif (PAM) and
(C) The PAM comprises a sequence selected from the group consisting of SEQ ID NO: 5512-5526 or SEQ ID NO: 5527-5537.
Method. The method of claim 149, wherein the double-stranded deoxyribonucleic acid polynucleotide comprises a first strand comprising a sequence complementary to the sequence of the engineered guide ribonucleic acid structure and a second strand comprising PAM. .. 15. The method of claim 151, wherein the PAM is directly adjacent to the 3'end of the sequence complementary to the sequence of the engineered guide ribonucleic acid structure. The Class 2 Type II Cas endonucleases are Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease. , Or the method of any one of claims 149-152, which is not a Cas13d endonuclease. The method according to any one of claims 149-153, wherein the class 2 type II Cas endonuclease is derived from a refractory microorganism. The double-stranded deoxyribonucleic acid polynucleotide according to any one of claims 149-154, which is a double-stranded deoxyribonucleic acid polynucleotide of eukaryote, plant, fungus, mammal, rodent, or human. the method of. a) The PAM comprises a sequence selected from the group consisting of SEQ ID NO: 5512-5515 and SEQ ID NO: 5527-5530.
b) The PAM comprises SEQ ID NO: 5516 or SEQ ID NO: 5531.
c) The PAM comprises SEQ ID NO: 5539.
d) The PAM comprises SEQ ID NO: 5517 or SEQ ID NO: 5518.
e) The PAM comprises SEQ ID NO: 5519.
f) The PAM comprises SEQ ID NO: 5520 or SEQ ID NO: 5535.
g) The PAM comprises SEQ ID NO: 5521 or SEQ ID NO: 5536.
h) The PAM comprises SEQ ID NO: 5522.
i) The PAM comprises SEQ ID NO: 5523 or SEQ ID NO: 5537.
j) The PAM comprises SEQ ID NO: 5524.
k) The PAM comprises or comprises SEQ ID NO: 5525.
l) The PAM comprises SEQ ID NO: 5526.
The method according to any one of claims 149-155. A method of modifying a target nucleic acid locus, wherein the method delivers any of the engineered nuclease systems according to any one of claims 1-129 to the target nucleic acid locus. Containing, the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure, wherein the complex is such that the complex binds to the target nucleic acid locus. A method in which the complex is configured to modify the target nucleic acid locus. 156. The method of claim 156, wherein modifying the target nucleic acid locus comprises binding, nicking, cleaving, or labeling the target nucleic acid locus. The method of any of claims 156-158, wherein the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 159. The method of claim 159, wherein the target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. The method of any one of claims 156-160, wherein the target nucleic acid locus is in vitro. The method according to any one of claims 156-160, wherein the target nucleic acid locus is intracellular. 16. The method of claim 162, wherein the cell is a prokaryotic cell, a bacterial cell, a eukaryotic cell, a fungal cell, a plant cell, an animal cell, a mammalian cell, a rodent cell, a primate cell, or a human cell. The step of delivering the engineered nuclease system to the target nucleic acid locus comprises delivering the nucleic acid of any of claims 135-140 or the vector of any of claims 142-146. The method according to any one of claims 162-163. 162-1. Method. 164. The method of claim 164, wherein the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked. One of claims 162-163, wherein the step of delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a capped mRNA comprising the open reading frame encoding the endonuclease. The method described in one. The method of any one of claims 162-163, wherein the step of delivering the engineered nuclease system to the target nucleic acid locus comprises delivering the translated polypeptide. The step of delivering the engineered nuclease system to the target nucleic acid locus delivers deoxyribonucleic acid (DNA) encoding the engineered guide ribonucleic acid structure operably linked to the ribonucleic acid (RNA) pol III promoter. The method of any one of claims 162-163, comprising: The method of any one of claims 156-169, wherein the endonuclease causes single-strand breaks or double-strand breaks at or proximal to the target nucleic acid locus.

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