JP2023179468A - Enzymes with ruvc domains - Google Patents

Abstract

To provide an engineered nuclease composition.SOLUTION: In one embodiment, an engineered nuclease composition comprises (a) an endonuclease, comprising a RuvC_III domain that comprises a sequence with at least 90% sequence identity to a specific sequence and (b) an engineered guide ribonucleic acid structure configured to form a complex with the endonuclease, wherein the engineered guide ribonucleic acid structure comprises a guide ribonucleic acid sequence configured to hybridize with a target deoxyribonucleic acid sequence.SELECTED DRAWING: Figure 1

Description

CROSS REFERENCES This application is a reference to U.S. Provisional Application No. 62/805,868, filed on February 14, 2019, entitled "MG1 ENZYMES WITH RUVC DOMAINS" and " U.S. Provisional Application No. 62/874,414 entitled “MG1 ENZYMES WITH RUVC DOMAINS” and U.S. Provisional Application No. 62/874,414 entitled “MG2 ENZYMES CONTAINING RUVC DOMAINS” filed on February 14, 2019. No. 805,878, and U.S. Provisional Application No. 62/805,899, filed February 14, 2019, entitled "MG3 ENZYMES WITH RUVC DOMAINS," each of which is incorporated by reference. Fully incorporated herein.

Cas enzymes, along with their associated clustered regularly spaced short palindromic repeats (CRISPR)-guided ribonucleic acids (RNAs), are widespread in prokaryotic immune systems (~45% of bacteria, ~84% of archaea). ) and appears to play a role in protecting such microorganisms from infectious viruses and non-self nucleic acids such as plasmids by CRISPR-RNA guided nucleic acid cleavage. Although the deoxyribonucleic acid (DNA) elements encoding CRISPR RNA elements may be relatively conserved in structure and length, their CRISPR-associated (Cas) proteins are highly diverse and interact with a wide variety of nucleic acids. Contains an action domain. Although CRISPR DNA elements have been observed as early as 1987, the programmable endonuclease cleavage capabilities of the CRISPR/Cas complex have been recognized relatively recently and have been shown to be useful in a variety of DNA manipulation and gene editing applications. This 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. The ASCII copy was created on February 13, 2020 and is 55921-703_601_SL. The file name is txt and the size is 23,363,113 bytes.

In some aspects, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system is: (a) an endonuclease comprising a RuvC_III domain and a HNH domain, wherein the endonuclease comprises: derived from an uncultivated microorganism, wherein the endonuclease is a class 2 type II Cas endonuclease; (b) configured to form a complex with the endonuclease; an engineered guide ribonucleic acid structure comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; (ii) an endonuclease as described above; an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind. In some embodiments, the RuvC_III domain comprises a sequence that has 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 aspects, the disclosure provides an engineered nuclease system, wherein the engineered nuclease system: (a) has 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 said endonuclease, said engineered guide ribonucleic acid structure comprising: (i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) a tracr ribonucleic acid sequence configured to bind to the endonuclease.

In some aspects, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system is configured to: (a) bind to a protospacer adjacent motif (PAM) sequence comprising SEQ ID NOs: 5512-5537; (b) an endonuclease configured to form a complex with the endonuclease, wherein the endonuclease is a class 2 type II Cas endonuclease; wherein the engineered guide ribonucleic acid structure comprises: (i) a guide ribonucleic acid sequence configured to hybridize to the target deoxyribonucleic acid sequence; (ii) configured to bind to the endonuclease. and an engineered guide ribonucleic acid structure comprising an assembled tracr ribonucleic acid sequence.

In some embodiments, the endonuclease is derived from a difficult-to-cultivate microorganism. In some embodiments, the endonuclease is not engineered to bind to different PAM sequences. In some embodiments, the endonuclease is Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13 c endonuclease , or not Cas13d endonuclease. In some embodiments, the endonuclease has less than 80% identity to Cas9 endonuclease. In some embodiments, the endonuclease further comprises an HNH domain. In some embodiments, the tracr ribonucleic acid sequences have at least 80% sequence identity over about 60-90 contiguous nucleotides selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. Contains sequences with specific characteristics.

In some aspects, the present disclosure provides an engineered nuclease system, wherein the engineered nuclease system comprises (a) an engineered guide ribonucleic acid structure, wherein the engineered guide ribonucleic acid structure comprises: (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 configured to: 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; and (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 adjacent motif (PAM) sequence selected from the group comprising SEQ ID NOs: 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 that includes a guide ribonucleic acid sequence and a tracr ribonucleic acid sequence.

In some embodiments, the guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence. In some embodiments, the guide ribonucleic acid sequence is 15-24 nucleotides in length. In some embodiments, the endonuclease includes one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS includes a sequence selected from SEQ ID NOs: 5597-5612.

In some embodiments, the engineered nuclease system has a first homology arm comprising, 5' to 3', a sequence of at least 20 nucleotides 5' of the target deoxyribonucleic acid sequence; Further included is 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 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 from separate 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 Verrucomicrobia, Candidatus Peregrinibacteria, or Candidatus Melinabacteria. In some embodiments, the endonuclease is derived from a bacterium that includes a 16S rRNA gene that has at least 90% identity to any one of SEQ ID NOs: 5592-5595.

In some embodiments, the HNH domain comprises a sequence that has 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 thereto. 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: 1827-1830 or SEQ ID NO: 1827-2140. include.

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: 3638-3641 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 NOs: 5615-5632. 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: 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 predicted to include a hairpin consisting of a stem and a loop, wherein the stem is a ribon of at least 10, at least 12, or at least 14 base pairs. nucleotides, and an asymmetric bulge within 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 comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO:1827; :5461 or SEQ ID NO:5476; and (c) the endonuclease 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 endonuclease comprises SEQ ID NO:5512 or SEQ ID NO:5527. configured to couple to a PAM containing a PAM. 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 comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 1828; : 5462 or SEQ ID NO: 5477; and (c) the endonuclease 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; configured to couple to a PAM containing a PAM. 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: 1829; (b) the guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: : 5463 or SEQ ID NO: 5478; and (c) the endonuclease comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to at least one of SEQ ID NO: 5463 or SEQ ID NO: 5478; 5529. 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:1830; (b) the guide RNA structure comprises a sequence that is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: : 5464 or SEQ ID NO: 5479; and (c) the endonuclease 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; 5530.

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: 2141-2142 or SEQ ID NO: 2141-2241. include. In some embodiments, the endonuclease produces a sequence that is at least 70%, 80%, or 90% identical to a sequence selected from the group consisting of SEQ ID NO: 3955-3956 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 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. include. In some embodiments, the guide RNA structure is at least 70%, 80%, or Contains sequences that are 90% identical. In some embodiments, the guide RNA structure comprises a tracr ribonucleic acid sequence that includes a hairpin comprising at least 8, at least 10, or at least 12 base pairs of 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2141; (b) the guide RNA structure comprises SEQ ID NO: 5490. and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO:5531. In some embodiments, (a) the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2142; (b) the guide RNA structure comprises SEQ ID NO: 5465. or (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 NOs: 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 predicted to include a hairpin with an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence. tracr ribonucleic acid sequence, wherein the tracr ribonucleic acid sequence includes, from 5' to 3', a first hairpin and a second hairpin, wherein the first hairpin is longer than the second hairpin. Has a long stem.

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: 2242-2244 or SEQ ID NO: 2247-2249. include. In some embodiments, the endonuclease produces 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. 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 NOs: 5639-5648. In some embodiments, the endonuclease produces 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. 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 predicted to include a hairpin with an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence. tracr ribonucleic acid sequence, wherein the tracr ribonucleic acid sequence includes, from 5' to 3', a first hairpin and a second hairpin, wherein the first hairpin is longer 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2247; (b) the guide RNA structure comprises SEQ ID NO: 5500. 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2248; (b) the guide RNA structure comprises SEQ ID NO: 5501. 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2249; (b) the guide RNA structure comprises SEQ ID NO: 5502. 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 the 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 SEQ ID NO: 432 or a sequence selected from the group consisting of 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 comprising a sequence selected from the group consisting of SEQ ID NO:5519. In some embodiments, (a) the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2253; (b) the guide RNA structure comprises SEQ ID NO: 5468. or comprising 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 a 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 a 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 a sequence selected from the group consisting of SEQ ID NOs: 4304-4312. 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 a 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2499; (b) the guide RNA structure comprises SEQ ID NO: 5469. or comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5505; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5520 or SEQ ID NO: 5535. configured.

In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2751 or a sequence selected from the group consisting of 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 NOs: 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2751; (b) the guide RNA structure comprises SEQ ID NO: 5470. or comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5506; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5521 or SEQ ID NO: 5536. configured.

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 SEQ ID NO: 4728 or a sequence selected from the group consisting of 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% relative to a 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 predicted to include at least two hairpins comprising ribonucleotides of less than 5 base pairs. In some embodiments, the endonuclease is configured to bind to a PAM comprising SEQ ID NO:5522. In some embodiments, (a) the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 2914; (b) the guide RNA structure comprises SEQ ID NO: 5471. or comprising 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 SEQ ID NO: 3175 or a sequence selected from the group consisting of 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 NOs: 5679-5686. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1354 or a sequence selected from the group consisting of 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3175; (b) the guide RNA structure comprises SEQ ID NO: 5472. or comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5508; and (c) the endonuclease is configured to bind to a PAM comprising SEQ ID NO: 5523 or SEQ ID NO: 5537. configured.

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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3331; (b) the guide RNA structure comprises SEQ ID NO: 5473. or comprising 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 SEQ ID NO: 5291 or a sequence selected from the group consisting of 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: 5694-5699. In some embodiments, the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1656 or a sequence selected from the group consisting of 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3475; (b) the guide RNA structure comprises SEQ ID NO: 5474. or (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 SEQ ID NO: 5390 or a sequence selected from the group consisting of 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 SEQ ID NO: 1756 or a sequence selected from the group consisting of 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 comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3569; (b) the guide RNA structure comprises SEQ ID NO: 5475. or comprising 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 the Smith-Waterman homology search algorithm. In some embodiments, sequence identity is determined using the parameters of a BLOSUM62 scoring matrix that sets a wordlength (W) of 3, an expectation (E) of 10, and a gap cost of 11 existence, 1 extension. as determined by the BLASTP homology search algorithm, as well as using the conditional compositional score matrix adjustment.

In some aspects, the present disclosure provides engineered guide ribonucleic acid polynucleotides, the engineered guide ribonucleic acid polynucleotides comprising: (a) a nucleotide sequence complementary to a target sequence in a target DNA molecule; (b) a protein binding segment comprising two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex, wherein the two complementary stretches of nucleotides hybridize to form a double-stranded RNA (dsRNA) duplex; stretches are covalently linked to each other with intervening nucleotides, wherein the engineered guide ribonucleic acid polynucleotides have RuvC_III domains having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637. and is configured to form a complex with an endonuclease comprising a target DNA molecule and target the complex to a target sequence of a target DNA molecule. In some embodiments, the DNA targeting segment is located 5' of both two complementary stretches of nucleotides.

In some embodiments, (a) the protein binding segment is at least 70%, at least 80%, or at least 90% relative to a sequence selected from the group consisting of SEQ ID NO: 5476-5479 or SEQ ID NO: 5476-5489. % identity 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; (c) the protein binding segment has at least 70%, at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5498-5499; (d) the protein binding segment has at least 70% identity to a sequence selected from the group consisting of SEQ ID NO: 5495-5497 and SEQ ID NO: 5500-5502; %, at least 80%, or at least 90%; (e) the protein binding segment has 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 the sequence (h) the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5505; (i) the protein binding segment comprises a sequence that has 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 is at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 5510; or (m) the protein binding segment comprises a sequence that has at least 70%, at least 80%, or at least 90% identity to SEQ ID NO:5511.

In some embodiments, (a) the guide ribonucleic acid polynucleotide comprises an RNA sequence that includes a hairpin that includes a stem and a loop, wherein the stem has at least 10, at least 12, or at least 14 base pairs. (b) the guide ribonucleic acid polynucleotide is expected to include a hairpin comprising at least 8, at least 10, or at least 12 base pairs of ribonucleotides; tracr ribonucleic acid sequence; (c) the guide ribonucleic acid polynucleotide comprises a hairpin having an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence; comprising a predicted guide ribonucleic acid sequence, wherein said tracr ribonucleic acid sequence comprises, 5' to 3', a first hairpin and a second hairpin, wherein said first hairpin comprises said first hairpin. or (d) the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence predicted to include at least two hairpins comprising ribonucleotides of less than 5 base pairs.

In some aspects, the present disclosure provides deoxyribonucleic acid polynucleotides encoding any of the engineered guide ribonucleic acid polynucleotides described herein.

In some aspects, the present disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid comprises a class 2 RuvC_III domain and a HNH domain. , wherein the class 2 type II Cas endonuclease is derived from a difficult-to-cultivate microorganism.

In some aspects, the present disclosure provides a nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein the nucleic acid comprises any one of SEQ ID NOs: 1827-3637. It encodes an endonuclease that contains a RuvC_III domain with at least 70% sequence identity to one RuvC_III domain. 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: 5572-5591 or a variant thereof having at least 70% sequence identity thereto. In some embodiments, the endonuclease includes a sequence encoding one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. In some embodiments, the NLS includes a sequence selected from SEQ ID NOs: 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% (b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5576-5577; (c) the nucleic acid sequence has identity; (d) 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; (e) the nucleic acid sequence has at least 70%, 80%, or 90% identity to SEQ ID NO: 5582; (f) the nucleic acid sequence has 70%, 80%, or 90% identity; (g) 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; 70%, 80%, or 90% identity; alternatively, (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 aspects, 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 a HNH domain, wherein the endonuclease is Originates from

In some aspects, the present disclosure provides vectors 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, the engineered guide ribonucleic acid structure comprising: (a) a target; (b) a guide ribonucleic acid sequence configured to hybridize to the deoxyribonucleic acid sequence; and (b) a tracr ribonucleic acid sequence configured to bind to the endonuclease. In some embodiments, the vector is a plasmid, minicircle, CELiD, adeno-associated virus (AAV)-derived virion, or lentivirus.

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

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

In some aspects, the present disclosure provides methods for joining, cleaving, labeling, or modifying double-stranded deoxyribonucleic acid polynucleotides, the methods comprising: (a) a class 2 type II Cas endonuclease; 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) wherein the double-stranded deoxyribonucleic acid polynucleotide includes a protospacer adjacent motif (PAM); (c) the PAM is from SEQ ID NO: 5512-5526 or SEQ ID NO: 5527-5537. including sequences selected from the group. In some embodiments, the double-stranded deoxyribonucleic acid polynucleotide includes a first strand that includes a sequence that is complementary to the sequence of the engineered guide ribonucleic acid structure and a second strand that includes a PAM. In some embodiments, the PAM is directly adjacent to the 3' end of a sequence that is complementary to the sequence of the engineered guide ribonucleic acid structure.

In some embodiments, the class 2 type II Cas endonuclease is Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease, or Cas13d endonuclease. In some embodiments, the class 2 type II Cas endonuclease is derived from a refractory microorganism. In some embodiments, the double-stranded deoxyribonucleic acid polynucleotide is a eukaryotic, plant, fungal, mammalian, rodent, or human double-stranded deoxyribonucleic acid polynucleotide.

In some embodiments, (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: (c) PAM includes SEQ ID NO: 5539; (d) PAM includes SEQ ID NO: 5517 or SEQ ID NO: 5518; (e) PAM includes SEQ ID NO: 5519; (f) PAM includes SEQ ID NO: 5519; SEQ ID NO: 5520 or SEQ ID NO: 5535; (g) PAM includes SEQ ID NO: 5521 or SEQ ID NO: 5536; (h) PAM includes SEQ ID NO: 5522; (i) PAM includes SEQ ID NO: 5523 or (j) PAM comprises SEQ ID NO: 5524; (k) PAM comprises SEQ ID NO: 5525; or (l) PAM comprises SEQ ID NO: 5526.

In some aspects, the present disclosure provides a method for modifying a target nucleic acid locus, the method comprising applying any of the engineered nuclease systems described herein to the target nucleic acid locus. the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure, wherein the complex is configured to form a complex upon binding of the complex to the target nucleic acid locus. , the complex is configured to modify a target nucleic acid locus. In some embodiments, modifying the target nucleic acid locus includes binding, nicking, cutting, 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, bacterial, eukaryotic, fungal, plant, animal, mammalian, rodent, primate, or human cell.

In some embodiments, 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. including doing. In some embodiments, delivering the engineered nuclease system to the target nucleic acid locus includes delivering a nucleic acid that includes an open reading frame encoding the endonuclease. In some embodiments, the nucleic acid includes 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 capped mRNA containing an open reading frame encoding the endonuclease. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises delivering translated polypeptide. In some embodiments, the engineered nuclease system to the target nucleic acid locus comprises deoxyribonucleic acid (DNA) encoding an engineered guide ribonucleic acid structure operably linked to a ribonucleic acid (RNA) pol III promoter. including delivering. In some embodiments, the endonuclease causes a single-stranded break or a double-stranded break at or proximal to the target locus.

Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure are shown and described. As will be understood, this disclosure is capable of other and different embodiments, and its various details may be modified in various obvious respects, all without departing from this disclosure. can. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. , incorporated herein by reference to the same extent.

The novel features of the invention are pointed out inter alia in the appended claims. A better understanding of the features and advantages of the present invention may be obtained from the following detailed description, which describes illustrative embodiments in which the principles of the invention may be employed, and the accompanying drawings, hereinafter referred to as "Figures" and "Figures". FIG.

Typical organization of various classes and types of CRISPR/Cas loci is shown. Figure 2 shows the structure of a natural class 2/type II crRNA/tracrRNA pair compared to a hybrid sgRNA to which both are linked. A conceptual diagram showing the organization of the CRISPR locus encoding enzymes from the MG1 family is shown. A conceptual diagram showing the organization of CRISPR loci encoding enzymes from the MG2 family is shown. A conceptual diagram showing the organization of CRISPR loci encoding enzymes from the MG3 family is shown. Figure 2 shows a structure-based alignment of the enzyme of the present disclosure (MG1-1) to Cas9 from Staphylococcus aureus (SEQ ID NO: 5613). Figure 2 shows a structure-based alignment of the enzyme of the present disclosure (MG2-1) to Cas9 from Staphylococcus aureus (SEQ ID NO: 5613). Figure 3 shows a structure-based alignment of the enzyme of the present disclosure (MG3-1) to Cas9 (SEQ ID NO: 5614) from Actinomyces naeslundii. Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). Figure 2 shows a structure-based alignment of MG1 family enzymes MG1-1 to MG1-6 (SEQ ID NOs: 5, 6, 9, 1, 2, and 3). In vitro cleavage of DNA by MG1-4 in complex with its corresponding sgRNA containing targeting sequences of various lengths is shown. Cellular cleavage of E. coli genomic DNA using MG1-4 and its corresponding sgRNA is shown. Dilution series of cells transformed with MG1-4 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. of HEK cells using the MG1-4 or MG1-6 constructs described in Example 11 together with their corresponding sgRNAs containing a variety of different targeting sequences targeting various locations in the human genome. Figure 2 shows cell indel formation generated by transfection. Figure 3 shows in vitro cleavage of DNA by MG3-6 in complex with its corresponding sgRNA containing targeting sequences of various lengths. Cellular cleavage of E. coli genomic DNA using MG3-7 and its corresponding sgRNA is shown. Dilution series of cells transformed with MG3-7 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 constructs of MG3-7 described in Example 13, along with their corresponding sgRNAs containing a variety of different targeting sequences targeting various locations in the human genome. Figure 2 shows the formation of cell indels. In vitro cleavage of DNA by MG15-1 in complex with its corresponding sgRNA containing targeting sequences of various lengths. Agarose gel is shown showing the results of PAM vector library cleavage in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Agarose gel is shown showing the results of PAM vector library cleavage in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Agarose gel is shown showing the results of PAM vector library cleavage in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Agarose gel is shown showing the results of PAM vector library cleavage in the presence of TXTL extracts containing various MG family nucleases and their corresponding tracrRNA or sgRNA. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. Figure 2 shows the predicted structure (eg, predicted as in Example 7) of the corresponding sgRNA of the MG enzyme described herein. FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). FIG. 6 shows a seqLogo representation of a PAM sequence derived via NGS as described herein (e.g., as described in Example 6). Cellular cleavage of E. coli genomic DNA using MG2-7 and its corresponding sgRNA is shown. Dilution series of cells transformed with MG2-7 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. Cellular cleavage of E. coli genomic DNA using MG14-1 and its corresponding sgRNA is shown. Dilution series of cells transformed with MG14-1 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. Cellular cleavage of E. coli genomic DNA using MG15-1 and its corresponding sgRNA is shown. Dilution series of cells transformed with MG15-1 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 THE SEQUENCE LISTING The Sequence Listing filed herewith provides exemplary polynucleotide and polypeptide sequences for use 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 shows 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 shows the peptide of the HNH domain of the above-mentioned MG1 nuclease.

SEQ ID NOs: 5476-5479 indicate the nucleotide sequences of MG1 tracrRNAs derived from the same loci as the above-mentioned MG1 nucleases (eg, the same loci as SEQ ID NOs: 1-4, respectively).

SEQ ID NOs: 5461-5464 indicate the nucleotide sequences of sgRNAs engineered to function with MG1 nuclease (eg, SEQ ID NOs: 1-4, respectively), where Ns represents the nucleotide of the targeting sequence.

SEQ ID NOs: 5572-5575 show the nucleotide sequences of E. coli codon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs: 1-4).

SEQ ID NOs: 5588-5589 show the nucleotide sequences of the human codon-optimized coding sequences for MG1 family enzymes (SEQ ID NOs: 1 and 3).

SEQ ID NO: 5616-5632 shows the peptide motif characteristics of MG1 family 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 shows the peptide of the HNH domain of the above MG2 nuclease.

SEQ ID NOs: 5490-5494 show the nucleotide sequences of MG2 tracrRNAs derived from the same loci as the MG2 nucleases described above (eg, the same loci as SEQ ID NOs: 320, 321, 323, 325, and 326, respectively).

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

SEQ ID NOs: 5572-5575 show the nucleotide sequences of E. coli codon-optimized coding sequences for MG2 family enzymes.

SEQ ID NO:5631-5638 shows the 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 NOs: 4056-4066 indicate the peptides of the HNH domain of the above MG3 nuclease.

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

SEQ ID NOs: 5466-5467 show the nucleotide sequences of sgRNAs engineered to function with MG3 nuclease (eg, SEQ ID NOs: 421-423).

SEQ ID NOs: 5578-5580 show the nucleotide sequences of E. coli codon-optimized coding sequences for MG3 family enzymes.

SEQ ID NO: 5639-5648 shows the 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 shows the peptide of 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 MG4 nuclease described above.

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

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

MG6

SEQ ID NO: 661-668 shows 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 shows the peptide of the HNH domain of the above MG3 nuclease.

MG7

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

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

SEQ ID NOs: 4304-4312 show the peptides 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 the MG7 nuclease described 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 shows the peptide of the HNH domain of the MG14 nuclease described above.

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

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

SEQ ID NO: 5650-5667 shows the 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 shows the peptide sequence of the RuvC_III domain of the above MG15 nuclease.

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

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

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

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

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

MG16

SEQ ID NO: 1093-1353 shows 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 shows the peptide of 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 described above.

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

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

SEQ ID NO: 5676-5678 shows 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 shows the peptide of 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 MG18 nuclease described above.

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

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

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

MG21

SEQ ID NO: 1512-1655 shows 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 shows the peptide of 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 MG21 nuclease described above.

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

SEQ ID NO: 5585 shows the nucleotide sequence of the E. coli codon-optimized coding sequence for MG21 family 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 shows the peptide sequence of the RuvC_III domain of the above MG22 nuclease.

SEQ ID NO: 5291-5389 shows the peptide of 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 MG22 nuclease described above.

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

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

SEQ ID NO: 5694-5699 shows the peptide sequence characteristics of MG22 family 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 NOs: 5390-5460 indicate the peptides of the HNH domain of the above-mentioned MG23 nuclease.

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

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

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

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

While various embodiments of the invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions may be devised by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized.

The practice of some of the methods disclosed herein may involve techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, unless otherwise specified. Make use of it. For example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biolo gy (F.M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc. ), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Application s, 6th Edition (R.I. Freshney, ed. (2010))) (which are incorporated herein by reference in their entirety). (incorporated into the specification).

As used herein, the singular forms "a," "an," and "the" refer to the singular forms "a," "an," and "the," unless the context clearly dictates otherwise. It is intended to include plural forms as well. Additionally, the terms "including," "includes," "having," "has," "with," or variations thereof, To the extent used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner analogous to the term "comprising". There is.

The term "about" or "approximately" means within an acceptable error range of a particular value, as determined by one of ordinary skill in the art, and that error range is based on how that value is measured or determined, i.e. depends in part on the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation 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 may be the basic structural, functional, and/or biological unit of an organism. A cell may originate from any organism that has one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-celled eukaryotic cells, protozoan cells, plant cells (e.g., crops, fruits, vegetables). , cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, cells of lycophytes, hornworts, liverworts, mosses), algal cells (e.g. Botryococcus braunii, Chlamydomonas reinhardti, Nannochloropsis gaditana, Chlorella pyrenoids) a, Sargassum patens C. Agardh etc.), seaweeds (e.g. kelp), fungal cells (e.g. , yeast cells, cells from mushrooms), animal cells, cells of invertebrates (e.g. Drosophila, cnidarians, echinoderms, nematodes, etc.), vertebrates (e.g. fish, amphibians, reptiles, birds, mammals) Examples include cells of mammals (eg, pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not have natural biological origin (for example, cells may be synthetically created and are 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. A nucleotide may be a monomeric unit of a nucleic acid sequence, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The terms nucleotide include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphate (GTP). , for example, dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide, as used herein, may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. The nucleotides may be unlabeled or may be detectably labeled, such as using a moiety that includes an optically detectable moiety (eg, a fluorophore). Labeling may also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine. (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL) ), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [ 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] ddTTP; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dC available from Amersham (Arlington Heights, Ill.) TP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP; from Boehringer Mannheim (Indianapolis, Ind.) 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 Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BO, available from Molecular Probes (Eugene, Oreg.) DIPY-TMR-14-dUTP , BODIPY -TR -14 -UTP, BODIPY -TR -14 -DUTP, CASCADE BLUE -7 -UTP, CASCADE BLUE -7 -DUTP, Fluoresane -12 -UTP, Fluoresane -12 -DUTP, OREGON GREEN 4 88-5 -DUTP , Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine 6-UTP, Tetramethylrhodamine 6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP can be mentioned. Nucleotides can also be labeled or marked by chemical modification. The chemically modified single nucleotide may be a biotin dNTP. Some non-limiting examples of biotinylated dNTPs include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin- 14-dCTP), and biotin-dUTP (eg, biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

The terms "polynucleotide," "oligonucleotide," and "nucleic acid" generally refer to nucleotides of any length, either in single-stranded, double-stranded, or multi-stranded form. (either deoxyribonucleotides or ribonucleotides), or analogs thereof. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide is , may exist in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may be any A polynucleotide may also have a three-dimensional structure and perform any function. A polynucleotide may include one or more analogs (e.g., modified backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, locked Nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g. rhodamine or fluorescein attached to sugars), thiol containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogs ), CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosine, and wyosine. Non-limiting examples of polynucleotides include genes or Coding or non-coding regions of fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), small molecules hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, Included are cell-free polynucleotides, including cell-free DNA (cfDNA), and cell-free RNA (cfRNA), nucleic acid probes, and primers. A sequence of nucleotides may be interrupted by non-nucleotide components.

The terms "transfection" or "transfected" generally refer to the introduction of a nucleic acid into a cell by non-viral or viral-based methods. A nucleic acid molecule can be a genetic sequence encoding a complete protein or 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 generally used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bonds. The term does not imply a specific length of the polymer, nor does it imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. not intended. This term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (eg, domains). The term also encompasses amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, oxidation, and other manipulations, such as conjugation with labeling moieties. 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 can include natural and unnatural amino acids, which are chemically modified to include groups or chemical moieties on the amino acid that do not occur naturally. 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 a nucleic acid or polypeptide sequence that is not found in naturally occurring nucleic acids or proteins. Non-natural may refer to affinity tags. Non-natural can refer to fusion. Non-naturally occurring may refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions, and/or deletions. Non-natural sequences include activities that may be exhibited by the nucleic acid and/or polypeptide sequences to which they are fused (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). ) and/or may be coded. A non-naturally occurring nucleic acid or polypeptide sequence can be genetically linked to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to encode a chimeric nucleic acid and/or a chimeric nucleic acid and/or polypeptide. may produce polypeptide sequences that

The term "promoter" as used herein generally refers to a regulatory DNA region that controls the transcription or expression of a gene and that is adjacent to or overlaps the nucleotide or region of nucleotides at which RNA transcription is initiated. It may be located at A promoter may contain specific DNA sequences that bind protein factors, often called transcription factors, that facilitate binding of RNA polymerase to the DNA and cause 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 an operably linked polynucleotide. Eukaryotic basic promoters typically, but not necessarily, contain a TATA box and/or a CAAT box.

The term "expression," as used herein, generally refers to the process by which a nucleic acid sequence or polynucleotide is transcribed (such as into mRNA or other RNA transcripts) from a DNA template, and/or Refers to the process by which the expressed mRNA is then 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 involve splicing of the mRNA in eukaryotic cells.

As used herein, "operably linked," "operably linked," or "operably linked," or grammatical equivalents thereof, generally refer to genetic elements, e.g. Refers to the juxtaposition of promoters, enhancers, polyadenylation sequences, etc., in which these elements are in a relationship that allows them to operate in a predicted manner. For example, a regulatory element, which may include a promoter and/or enhancer sequence, is operably linked to a coding region if the regulatory element assists in initiating transcription of the coding sequence. There may be intervening residues between the regulatory element and the coding region so long as this functional relationship is maintained.

"Vector," as used herein, generally refers to a macromolecule or association of macromolecules that includes or is associated with a polynucleotide and that directs the transfer of the polynucleotide into 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, such as regulatory elements, operably linked to the gene to promote expression of the gene in the target.

As used herein, "expression cassette" and "nucleic acid cassette" are generally used interchangeably 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 that of the full-length DNA or protein sequence. The biological activity of a DNA sequence may be its ability to affect expression in a manner known to be attributable to the full-length sequence.

As used herein, a "manipulated" subject generally indicates that the subject has been modified by human intervention. By way of non-limiting example, a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified such that the ligated product possesses a function not present in the original nucleic acid. The nucleic acid may be modified by ligation to a nucleic acid with which it is not naturally associated, such that the nucleic acid is associated with it in nature; an engineered nucleic acid may be synthesized in vitro with a sequence that does not occur in nature; A protein may be modified by changing its amino acid sequence to one that does not occur in nature; the engineered protein may gain new functions or properties. An “operated” system includes at least one operated component.

As used herein, "synthetic" and "artificial" refer to low sequence identity (e.g., less than 50% sequence identity, less than 25% sequence identity) to naturally occurring human proteins. , 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 terms "tracrRNA" or "tracr sequence," as used herein, generally refer to wild-type exemplary tracrRNA sequences (e.g., tracrRNA from S. pyogenes, Staphylococcus aureus, etc., or 5476-5511) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% sequence identity tracrRNA sequence may refer to a nucleic acid having a tracrRNA sequence similar to its wild-type exemplary tracrRNA sequence. tracrRNA is up to about 5%, 10%, 20%, 30%, 40%, 50%, relative to wild type exemplary tracrRNA sequences (e.g., 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 modified forms of tracrRNA that include nucleotide changes such as deletions, insertions, or substitutions, variants, mutations, or chimeras. When tracrRNA refers to a nucleic acid that is at least about 60% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from S. pyogenes, Staphylococcus aureus, etc.) sequence over a stretch of at least 6 contiguous nucleotides. There is. For example, a tracrRNA sequence is at least about 60% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least 6 contiguous nucleotides, at least about % 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 a genomic sequence by identifying regions that have complementarity to portions of repeat sequences in adjacent CRISPR arrays.

As used herein, "guide nucleic acid" may generally refer to a nucleic acid that is capable of hybridizing to another nucleic acid. The guide nucleic acid may be RNA. The guide nucleic acid may be DNA. A guide nucleic acid may be programmed to site-specifically bind to a sequence of nucleic acids. A targeted or target nucleic acid may include nucleotides. Guide nucleic acids may include nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes to a guide nucleic acid is sometimes referred to as the complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore may not be complementary to a guide nucleic acid may be referred to as a non-complementary strand. A guide nucleic acid may include one polynucleotide strand and may be referred to as a "single guide nucleic acid." A guide nucleic acid may include two polynucleotide strands and is sometimes referred to as a "double guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" is inclusive and may refer to both single and dual guide nucleic acids. A guide nucleic acid may include a segment, sometimes referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence." Nucleic acid targeting segments may include subsegments sometimes 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 the relationship between two (e.g., pairwise alignments) or more (e.g., multiple sequence alignments). Refers to sequences that are the same when compared or aligned for maximum correspondence over a local or global comparison window, as determined using a sequence comparison algorithm, or Having a specified percentage of the same amino acid residues or nucleotides. Sequence comparison algorithms suitable for polypeptide sequences use, for example, the parameters of the BLOSUM62 scoring matrix, which sets the gap cost at wordlength (W) of 3, expectation (E) of 10, and existence of 11, extension of 1. and BLASTP using conditional compositional score matrix adjustment for polypeptide sequences longer than 30 residues; wordlength (W) of 2, expectation (E) parameters of 1000000. , and 30 BLASTP using a PAM30 scoring matrix that sets the gap cost at 9 to open gaps for sequences with less than a residue and 1 to extend gaps (these are available at https://blast.ncbi.nlm (default parameters for BLASTP in the BLAST suite available at .nih.gov); CLUSTALW with parameters; Smith-Waterman homology search algorithm with parameters of match of 2, mismatch of -1, and gap of -1; Includes: MUSCLE with default parameters; MAFFT with parameters of retree of 2 and maximizations of 1000; Novafold with default parameters; HMMER hmmalign with default parameters.

As used herein, the term "RuvC_III domain" generally refers to the three RuvC endonuclease domains (the RuvC nuclease domain, which is composed of three discrete segments, RuvC_I, RuvC_II, and RuvC_III). Refers to discrete segments of the eye. RuvC domains or segments thereof are generally constructed by alignment to known domain sequences, structural alignment to proteins with annotated domains, or by hidden Markov models (HMMs) constructed based on known domain sequences (e.g., in RuvC_III). Pfam HMM PF18541).

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

overview

The discovery of new Cas enzymes with unique functions and structures offers the potential to further disrupt deoxyribonucleic acid (DNA) editing technologies, improving speed, specificity, functionality, and ease of use. I can do it. The predicted prevalence of clustered regularly spaced short palindromic repeat (CRISPR) systems in microorganisms and the functionally characterized CRISPR/CRISPR systems compared to a wide variety of microbial species. Cas enzymes are relatively absent from the literature. This is partly because a vast number of microbial species may not be easily cultured in laboratory conditions. Metagenomic sequencing from natural environmental niches representing many microbial species may present the potential to rapidly increase the number of known new CRISPR/Cas systems and facilitate the discovery of new oligonucleotide editing functions. A recent example of the usefulness of such approaches is shown by the 2016 discovery of the CasX/CasY CRISPR system from metagenomic analysis of natural microbial communities.

The CRISPR/Cas system is an RNA-directed nuclease complex that has been described to function as an adaptive immune system in microorganisms. In their natural context, CRISPR/Cas systems arise in CRISPR (Clustered Regularly Arranged Short Palindromic Repeats) operons or loci, which generally have two parts: (i) RNA-based targeting; an array of short repeat sequences (30-40 bp) separated by equally short spacer sequences encoding elements; (ii) encoding nuclease polypeptides, along with accessory proteins/enzymes, targeted by RNA-based targeting elements; ORF encoding Cas. Efficient nuclease targeting of a particular target nucleic acid sequence generally involves (i) complementary hybridization between the first 6 to 8 nucleic acids of the target (target seed) and a crRNA guide; ii) the presence of a protospacer adjacent motif (PAM) sequence within a defined vicinity of the target seed (PAM is generally a sequence that is not commonly represented within the host genome). Depending on the exact functionality and configuration of the system, CRISPR-Cas systems are generally divided into two classes, five types, and sixteen subtypes based on shared functional characteristics and evolutionary similarities. Be organized.

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

Type I CRISPR-Cas systems are considered to be of moderate complexity in terms of components. In type I CRISPR-Cas systems, an array of RNA-targeting elements is transcribed as long precursor crRNAs (pre-crRNAs), which are processed with repeating elements to release short, mature crRNAs, and this short, mature The crRNAs, when followed by an appropriate short consensus sequence called the protospacer adjacent motif (PAM), direct the nuclease complex to the nucleic acid target. This processing occurs through the endoribonuclease subunit (Cas6) of a large endonuclease complex called the cascade, which further includes the nuclease (Cas3) protein component of the crRNA-directed nuclease complex. Cas I nuclease functions primarily as a DNA nuclease.

Type III CRISPR systems may be characterized by the presence of a central nuclease known as Cas10, along with repeat-associated mysterious proteins (RAMPs) that include the Csm or Cmr protein subunits. As in type I systems, mature crRNA is processed from pre-crRNA using enzymes such as Cas6. Unlike type I and type II systems, type III systems appear to target and cleave DNA-RNA duplexes, such as the DNA strand used as a template for RNA polymerase.

The type IV CRISPR-Cas system involves two genes: the highly reduced large subunit nuclease (csf1) and the RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and in some cases , 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 single polypeptide multi-domain nuclease effectors and include types II, V, and VI.

Type II CRISPR-Cas systems are considered the simplest from a component standpoint. In type II CRISPR-Cas systems, CRISPR arrays are processed into mature crRNAs using specialized rather than small trans-encoded crRNAs (tracrRNAs) with complementary regions to the array repeats. The presence of an endonuclease subunit is required; tracrRNA interacts with both its corresponding effector nuclease (e.g., Cas9) and repeat sequences to form a precursor dsRNA structure, which tracrRNA and crRNA to produce a mature effector enzyme loaded with both tracrRNA and crRNA. Cas II nuclease is known as DNA nuclease. Type 2 effectors generally exhibit a structure consisting of a RuvC-like endonuclease domain that adopts an RNase H fold in which an unrelated HNH nuclease domain is inserted within the fold of the RuvC-like nuclease domain. The RuvC-like domain is responsible for cleavage of the target (eg, crRNA-complementary) DNA strand, while the HNH domain is responsible for cleavage of the displaced DNA strand.

Type V CRISPR-Cas systems are characterized by a nuclease effector (eg, Cas12) structure that is similar to that of type II effectors, including a RuvC-like domain. Similar to type II, most (but not all) type V CRISPR systems use tracrRNA to process pre-crRNA into mature crRNA; however, they cleave pre-crRNA into multiple crRNAs. Unlike the type II system, which requires RNAse III to cleave the pre-crRNA, the type V system can use the effector nuclease itself to cleave the pre-crRNA. Like 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 type V enzymes (e.g., Cas12a) generate robust single-stranded non-transferases that are activated by the first crRNA-directed cleavage of the double-stranded target sequence. It appears to have specific deoxyribonuclease activity.

The type VI CRIPSR-Cas system has RNA-guided RNA endonucleases. Instead of RuvC-like domains, single polypeptide effectors of type VI systems (eg, Cas13) contain two HEPN ribonuclease domains. Unlike both type II and type V systems, type VI systems do not appear to require tracrRNA to process pre-crRNA into crRNA. However, similar to type V systems, some type VI systems (e.g., C2C2) utilize a robust single-stranded, nonspecific nuclease activated by the first crRNA-directed cleavage of the target RNA. It appears to have (ribonuclease) activity.

Class II CRISPR-Cas has been most widely adopted for engineering and development as designer nuclease/genome editing applications due to its simpler structure.

One of the early adaptations of such a system for in vitro use was by Jinek et al. (Science. 2012 Aug 17;337(6096):816-2, which is fully incorporated herein by reference. (incorporated). In Jinek's test, (i) S. recombinantly expressed and purified full-length Cas9 (e.g., a type II Cas enzyme of class II) isolated from P. pyogenes SF370, (ii) the target DNA sequence desired to be cleaved; Purified mature ~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 a T7 promoter sequence); A system containing iii) purified tracrRNA transcribed in vitro from a synthetic DNA template with a T7 promoter sequence, and (iv) Mg2+ was first described. Jinek then described an improved engineered system in which ( The crRNA of ii) is joined to the 5' end of (iii) by a linker (eg GAAA) (compare upper and lower panels of Figure 2).

Mali et al. (Science. 2013 Feb 15; 339(6121):823-826.), which is fully incorporated herein by reference, then (i) added a C-terminal nuclear localization sequence ( with an ORF encoding a codon-optimized Cas9 (e.g., a type II Cas enzyme of class II) under a suitable mammalian promoter with an appropriate polyadenylation signal (e.g., TK pA signal); ii) an ORF encoding an sgRNA under a suitable polymerase III promoter (e.g. U6 promoter) (5' sequence starting with G followed by 20 nt of complementary targeting nucleic acid sequence and a 3' tracr binding sequence linked thereto) This system was adapted for use in mammalian cells by providing a DNA vector encoding a tracrRNA sequence, a linker, and a tracrRNA sequence.

MG1 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may include a RuvC_III domain, where the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 1827-2140. In some cases, the endonuclease may include a RuvC_III domain, where the RuvC_III domain is 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 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical. In some cases, the endonuclease may include a RuvC_III domain, where the endonuclease is substantially identical to any one of SEQ ID NOs: 1827-2140. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 1827-1831. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% of 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%, at least about 99% identity. There are cases. In some cases, the endonuclease includes a RuvC_III domain substantially identical to any one of SEQ ID NOs: 1827-1831. 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%, at least about 92% of SEQ ID NO: 1827. , at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity. 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%, at least about 92%, relative to SEQ ID NO: 1828. It may include RuvC_III domains 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%, at least about 99% identity. 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%, at least about 92%, relative to SEQ ID NO: 1829. It may include RuvC_III domains 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%, at least about 99% identity. 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%, at least about 92%, relative to SEQ ID NO: 1830. It may include RuvC_III domains 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%, at least about 99% identity. 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%, at least about 92%, relative to SEQ ID NO: 1831. It may include RuvC_III domains 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%, at least about 99% identity.

The endonuclease may include 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% of 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%. May include. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3955. The endonuclease may include 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% of 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%. May include. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3955. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3638-3641. 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% of any one of SEQ ID NOs: 3638-3641. %, 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%. May include. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 3638-3641. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NO:3638. 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%, HNH domains 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 substantially identical to any one of SEQ ID NO:3638. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NO:3639. 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%, HNH domains 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 substantially identical to any one of SEQ ID NO:3639. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NO:3640. 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%, HNH domains 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 substantially identical to any one of SEQ ID NO:3640. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NO:3641. 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%, HNH domains 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 substantially identical to any one of SEQ ID NO:3641.

In some cases, the endonuclease is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% of any one of SEQ ID NO: 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% , at least about 97%, at least about 98%, or at least about 99%. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NO: 1-6 or 9-319. In some cases, the endonuclease is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% of any one of SEQ ID NO: 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%. In some cases, the endonuclease is substantially identical to any one of SEQ ID NOs: 1-4. In some cases, the endonuclease may include a peptide motif substantially identical to any one of SEQ ID NOs: 5615, 5616, or 5617.

In some cases, the endonuclease may include a variant with one or more nuclear localization sequences (NLS). The NLS may 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 NO: 1-6 or 9-319, or at least about 30% to any one of SEQ ID NO: 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%, Added to the N-terminus or C-terminus of a variant with 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. Where 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 include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS may include any of the sequences in Table 1 below, or a combination thereof.

In some cases, the endonuclease may be recombinant (eg, cloned, expressed, and purified by suitable methods, such as expression in E. coli followed by epitope tag purification). In some cases, the endonuclease may be derived from a bacterium that has a 16S rRNA gene that has at least about 90% identity to any one of SEQ ID NOs: 5592-5595. The endonuclease is 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 a 16S rRNA gene that is at least about 98%, or at least about 99% identical. The endonuclease may be derived from a species having a 16S rRNA gene substantially identical to any one of SEQ ID NOs: 5592-5595. The endonuclease may be derived from bacteria belonging to the phylum Verrucomicrobia or Candidatus Peregrinibacteria.

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditions. may be determined by the BLASTP algorithm using a conditional compositional score matrix adjustment.

Optionally, the system described above comprises (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence. ) may be included. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, the 5' most nucleotide of the targeting region may be a G. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence can be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3': a non-natural guide nucleic acid sequence capable of hybridizing to a target sequence in a cell; and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a specific sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. at least about 80%. The tracr sequence has at least about 60 to 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) contiguous nucleotides of at least about 80%. In some cases, the tracrRNA is at least about 60-90 (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) contiguous nucleotides, 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 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 (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 contiguous nucleotides. tracrRNA may include any of SEQ ID NOs: 5476-5489.

Optionally, the 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 with a 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% of 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%. The sgRNA may include a sequence substantially identical to any one of SEQ ID NOs: 5461-5464.

In some cases, the above system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second site is It is 3' of part 1. In some cases, the above system includes at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) in the 5' to 3' of the first region. a first homology arm comprising a sequence of nucleotides; a synthetic DNA sequence of at least about 10 nucleotides; and a second homology arm comprising a sequence of 300, 500, or 1 kb) nucleotides.

In other aspects, the disclosure provides methods for modifying a target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein, including an enzyme and at least one synthetic guide RNA (sgRNA) disclosed herein, to a target nucleic acid locus. may be included. The enzyme may form a complex with at least one sgRNA and modify the target nucleic acid locus when the complex binds to the target nucleic acid locus. Delivering an enzyme to said genetic locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said genetic locus may include electroporating cells with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system with a nucleic acid comprising the locus of interest in a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. Enzymes may cause single-strand breaks or double-strand breaks at or near the target locus of interest.

If the target nucleic acid locus is likely to be intracellular, the enzyme may be at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease is any one of SEQ ID NOs: 5572-5575, or at least about 30% of any one of SEQ ID NOs: 5572-5575. %, 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%. may contain an array of Optionally, the nucleic acid includes a promoter to which an open reading frame encoding an endonuclease is operably linked. The promoter may be a 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. The at least one engineered sgRNA may be provided as deoxyribonucleic acid (DNA) containing a genetic sequence encoding said at least one engineered sgRNA operably linked to a 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 a human.

In some cases, the present disclosure may provide expression cassettes comprising the systems disclosed herein, or the nucleic acids described herein. In some cases, the expression cassette or nucleic acid may be provided as a vector. In some cases, expression cassettes, nucleic acids, or vectors may be provided in cells. In some cases, the cell is a bacterial cell having a 16S rRNA gene that has 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 that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may include a RuvC_III domain, where 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 include a RuvC_III domain, where the RuvC_III domain is at least about 20%, at least about 25%, at least about 30% relative 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% , having at least about 99% identity. In some cases, the endonuclease may include a RuvC_III domain and is substantially identical to any one of SEQ ID NOs: 2141-2142. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2141-2142. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% of 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% %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity. May include. In some cases, the endonuclease includes a RuvC_III domain substantially identical to any one of SEQ ID NOs: 2141-2142.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955-4055. 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% of 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% identical. There is. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 3955-4055. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 3955-3956. 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 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 substantially identical to any one of SEQ ID NOs: 3955-3956.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-420. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 320-321.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NOs: 320-420, or at least about 35%, at least about 40% relative to 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence has at least about 60 to 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) contiguous nucleotides of at least about 80%. In some cases, the tracrRNA is at least about 60-90 (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) contiguous nucleotides, 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 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 to 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 contiguous nucleotides. tracrRNA may include any of SEQ ID NOs: 5490-5494.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5465.

In some embodiments, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is 3' for the first region. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the locus of the target nucleic acid may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to any one of SEQ ID NOs: 5576-5577, or a sequence substantially identical to any one of SEQ ID NOs: 5576-5577. at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% %, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. May include. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG3 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2242-2251. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2242-2244. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2242-2244.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056-4066. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4066. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4056-4058. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4058.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-431. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 421-423.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NOs: 421-431, or at least about 35%, at least about 40% relative to any one of SEQ ID NOs: 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence has at least about 60 to 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) contiguous nucleotides of at least about 80%. In some cases, the tracrRNA is at least about 60-90 (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) contiguous nucleotides, 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 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 to 100 (e.g., 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 contiguous nucleotides. The tracrRNA may include any of SEQ ID NOs: 5495-5502.

Optionally, the 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: 5466-5467. may include an array 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% of 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%. The sgRNA may include a sequence substantially identical to any one of SEQ ID NOs: 5466-5467.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' to the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus can be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) containing the open reading frame encoding the endonuclease has a sequence substantially identical to any one of SEQ ID NOs: 5578-5580, or a sequence substantially identical to any one of SEQ ID NOs: 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%. May include. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG4 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2253-2481. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2253-2481. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2253-2481.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067-4295. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4067-4295. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4067-4295. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4067-4295.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 432-660.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 432-660, or at least about 35%, at least about 40% relative 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of SEQ ID NO: 5503. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5503. 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 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 to 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) of SEQ ID NO: 5503. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5503.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5468.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the locus of the target nucleic acid may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG6 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may include a RuvC_III domain, wherein the RuvC_III domain has at least about 70% sequence identity to any one of SEQ ID NOs: 2482-2489. 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2482-2489.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4296-4303. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4056-4066.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 661-668.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 661-668, or at least about 35%, at least about 40% relative 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%.

In some cases, the system may include two different guide RNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is 3' to the first region. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus can be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG7 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2490-2498. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2490-2498. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2490-2498.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304-4312. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4304-4312. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4304-4312. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4304-4312.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 669-677.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 669-677, or at least about 35%, at least about 40% relative 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 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) contiguous nucleotides of SEQ ID NO: 5504. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5504. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5504. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5504.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus can be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG14 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2499-2750. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2499-2750. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2499-2750.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313-4564. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4313-4564. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4313-4564. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4313-4564.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 678-929.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 678-929, or at least about 35%, at least about 40% relative 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 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) contiguous nucleotides of SEQ ID NO: 5505. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5505. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5505. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5505.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5469.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, at least about 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG15 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2751-2913. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2751-2913. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2751-2913.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565-4727. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4565-4727. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4565-4727. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4565-4727.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 930-1092.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 930-1092, or at least about 35%, at least about 40% relative to any one of SEQ ID NO: 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of SEQ ID NO: 5506. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5506. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5506. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5506.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5470.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, at least about 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG16 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 2914-3174. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 2914-3174. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 2914-3174.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728-4988. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4728-4988. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4728-4988. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4728-4988.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1093-1353.

In some cases, the endonuclease may include a variant 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 the N-terminus or C-terminus to any one of SEQ ID NOs: 1093-1353, or at least about 30%, at least about 35% to any one of SEQ ID NOs: 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%, Variants 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 may be added. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of SEQ ID NO: 5507. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5507. 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 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 to 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) of SEQ ID NO: 5507. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5507.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5471.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, at least about 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG18 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 comprise a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. In some cases, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 3175-3300. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3175-3300. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3175-3300.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989-5146. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4989-5146. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 4989-5146. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 4989-5146.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1354-1511.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 1354-1511, or at least about 35%, at least about 40% relative 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of SEQ ID NO: 5508. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5508. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5508. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5508.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5472.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' to the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, at least about 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG21 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 3331-3474. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3331-3474. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3331-3474.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147-5290. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5147-5290. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5147-5290. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5147-5290.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1512-1655.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 1512-1655, or at least about 35%, at least about 40% relative to any one of SEQ ID NO: 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 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) contiguous nucleotides of SEQ ID NO: 5509. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5509. 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 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 to 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) of SEQ ID NO: 5509. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5509.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5473.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' of the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the locus of the target nucleic acid may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG22 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 include a RuvC_III domain, wherein the RuvC_III domain is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 3475-3568. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3475-3568. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3475-3568.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291-5389. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5291-5389. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5291-5389. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5291-5389.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1656-1755.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 432-660, or at least about 35%, at least about 40% relative to any one of SEQ ID NO: 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of SEQ ID NO: 5510. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5510. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5510. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5510.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5474.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' to the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the locus of the target nucleic acid can be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

MG23 enzyme

In one aspect, the present disclosure provides an engineered nuclease system that includes (a) an endonuclease. In some cases, the endonuclease is a Cas endonuclease. In some cases, the endonuclease is a type II class II Cas endonuclease. The endonuclease may 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 about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99% identical. Optionally, the endonuclease may include a RuvC_III domain, wherein the endonuclease is substantially identical to any one of SEQ ID NOs: 3569-3637. The endonuclease may include a RuvC_III domain having at least about 70% sequence identity to any one of SEQ ID NOs: 3569-3637. In some cases, the endonuclease is at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about RuvC_III domains having an identity of 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. May contain. In some cases, the endonuclease may include a RuvC_III domain that is substantially identical to any one of SEQ ID NOs: 3569-3637.

The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390-5640. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5390-5460. The endonuclease may include an HNH domain having at least about 70% identity to any one of SEQ ID NOs: 5390-5460. 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 HNH domains having an identity of 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%. May contain. The endonuclease may contain an HNH domain substantially identical to any one of SEQ ID NOs: 5390-5460.

In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826. In some cases, the endonuclease is 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 Variants having 97%, at least about 98%, or at least about 99% identity may be included. In some cases, the endonuclease may be substantially identical to any one of SEQ ID NOs: 1756-1826.

In some cases, the endonuclease may include a variant 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% at the N-terminus or C-terminus of any one of SEQ ID NO: 1756-1826, or at least about 30%, at least about 35%, at least about 40% relative to any one of SEQ ID NO: 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% identity. The NLS may be an 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%, at least about 99% identity to any one of SEQ ID NOs: 5593-5608. I can do it. The NLS can include a sequence substantially identical to any one of SEQ ID NOs: 5593-5608. The NLS can include any of the sequences in Table 1, or a combination thereof:

In some cases, sequence identity may be determined by BLASTP, CLUSTALW, MUSCLE, MAFFT, Novafold, or Smith-Waterman homology search algorithms. Sequence identity was determined using the BLOSUM62 scoring matrix using parameters of wordlength (W) of 3, expectation (E) of 10, and gap cost with existence of 11, extension of 1, and conditional composition. It can be determined with the BLASTP algorithm using a score matrix adjustment.

Optionally, the system comprises: (b) at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease having a 5' targeting region complementary to the desired cleavage sequence; May include. In some cases, the 5' targeting region may include a PAM sequence that is compatible with an endonuclease. In some cases, most nucleotides 5' of the targeting region may be G's. In some cases, the 5' targeting region may be 15-23 nucleotides in length. The guide sequence and tracr sequence may be provided as separate ribonucleic acids (RNA) or as a single ribonucleic acid (RNA). The guide RNA may include a crRNA tracrRNA binding sequence 3' of the targeting region. The guide RNA may include a tracrRNA sequence preceded by a 4-nucleotide linker 3' of the crRNA tracrRNA binding region. The sgRNA may include, 5' to 3', a non-natural guide nucleic acid sequence capable of hybridizing to a cellular target sequence, and a tracr sequence. In some cases, the non-natural guide nucleic acid sequence and the tracr sequence are covalently linked.

In some cases, the tracr sequence may have a special sequence. A tracr sequence comprises at least about 60 to 100 (e.g., 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) contiguous nucleotides of a natural tracr RNA sequence. may have at least about 80%. The tracr sequence comprises at least about 60 to 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) contiguous nucleotides of SEQ ID NO: 5511. may have at least about 80% sequence identity to. In some cases, the tracrRNA is at least about 60 to 90 (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) of SEQ ID NO: 5511. 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 consecutive nucleotides %, at least about 97%, at least about 98%, or at least about 99%. In some cases, the tracrRNA comprises at least about 60 to 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) of SEQ ID NO: 5511. Consecutive nucleotides may be substantially identical. tracrRNA may include SEQ ID NO:5511.

Optionally, the at least one engineered synthetic guide ribonucleic acid (sgRNA) capable of forming a complex with an endonuclease may include 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 include sequences having at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity. The sgRNA may include a sequence substantially identical to SEQ ID NO:5475.

In some cases, the system may include two different sgRNAs that target a first region and a second region for cleavage at a target DNA locus, where the second region is a second region. 3' for a region of 1. In some cases, the system includes 5' to 3' at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) nucleotides 5' to the first region. a first homology arm comprising a synthetic DNA sequence of at least about 10 nucleotides, and 3′ of the second region at least about 20 (e.g., at least about 40, 80, 120, 150, 200, 300, 500, or 1 kb) of nucleotides).

In other aspects, the disclosure provides methods for modifying a desired target nucleic acid locus. The method comprises delivering any of the non-natural systems disclosed herein comprising an enzyme disclosed herein and at least one synthetic guide RNA (sgRNA) to a target nucleic acid locus. But that's fine. The enzyme may form a complex with at least one sgRNA and modify the desired target nucleic acid locus upon binding of the complex to the desired target nucleic acid locus. Delivering an enzyme to said locus may include transfecting a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to said locus may include electroporating a cell with said system or a nucleic acid encoding said system. Delivering a nuclease to the locus may include incubating the system in a buffer with a 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 within a eukaryotic or prokaryotic cell. The cell may be an animal cell, a human cell, a bacterial cell, an archaeal cell, or a plant cell. The enzyme may induce single-stranded or double-stranded breaks at or near the desired target locus.

In cases where the target nucleic acid locus may be intracellular, the enzyme has at least about 75% (e.g., at least about 90%, at least about 91%, at least Enzymes having RuvC_III domains having an 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 also be provided as a nucleic acid containing an encoding open reading frame. The deoxyribonucleic acid (DNA) comprising 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%, at least about 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 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 may be included. In some cases, the nucleic acid includes a promoter to which is operably linked an open reading frame encoding an endonuclease. The promoter may be a CMV, EF1a, SV40, PGK1, Ubc, human β-actin, CAG, TRE, or CaMKIIa promoter. The endonuclease may be provided 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 provided as deoxyribonucleic acid (DNA) comprising a genetic sequence encoding said at least one engineered sgRNA operably linked to a ribonucleic acid (RNA) pol III promoter. . In some cases, the organism may be eukaryotic. In some cases, the organism may be a fungus. In some cases, the organism may be a human.

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

Example 2A. -Discovery of the MG1 family of CRISPR systems Analysis of the data from the metagenomic analysis of Example 1 revealed that there were initially six members (MG1-1, MG1- recorded as SEQ ID NOs: 5, 6, 1, 2, and 3, respectively). A new cluster of previously undescribed putative CRISPR systems was revealed, including MG2, MG1-3, MG1-4, MG1-5, and MG1-6). This family is characterized by enzymes with HNH and RuvC domains. The RuvC domains of this family have a RuvC_III portion with low homology to previously described Cas9 family members. Although the original family members share up to 56.8% identity, all six enzymes have divergent RuvC_III portions of their RuvC domains, including RHHALDAMV (SEQ ID NO: 5615) and KHHALDAMC (SEQ ID NO: 5616). ), or KHHALDAIC (SEQ ID NO: 5617). These motifs are not found in other Cas9-like enzymes described. The protein and nucleic acid sequences corresponding to these new enzymes and their associated subdomains are presented in the sequence listing. The putative tracrRNA sequences were identified based on their relative position to other genes and are presented as SEQ ID NOs: 5476-5479. The enzyme system is based on the sequence of 16S rRNA from a genome bin containing a CRISPR system, and is based on Phylum Verrucomicrobia, Phylum Candidatus Peregrinibacteria, or Phylum Candidatus Melainabacteri. It is thought to originate from a. The sequences of 16S rRNA are presented as SEQ ID NOs: 5592-5596. Detailed domain-level alignments of CRISPR system sequences (Mol Cell. 2015 Nov 5;60(3):385-97) that evoke features described by Shmakov et al. Depicted in 9G and 9H. Comparison of MG1-1, 1-2, and 1-3 with additional proprietary protein datasets revealed an additional protein sequence with a similar architecture, presented as SEQ ID NO: 7-319. Ta. The sequences of these MG1 proteins led to the discovery of additional MG1 motifs as shown in SEQ ID NOs: 5618-5632.

Example 2B. -Discovery of the MG2 family of CRISPR systems Analysis of data from the metagenomic analysis of Example 1 shows that so far, including six members (MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6) A new cluster of putative CRISPR systems has been uncovered that has not yet been described. Protein and nucleic acid sequences corresponding to these new enzymes and exemplary subdomains are presented as SEQ ID NOs: 320, 322-325. Putative tracrRNA sequences were identified within the operon based on their relative positions to other genes and are presented as SEQ ID NOs: 5490, 5492-5494, and 5538. A detailed domain-level alignment of these sequence pairs for Cas9 is depicted in FIG. 7, as shown by Shmakov et al. (Mol Cell. 2015 Nov 5;60(3):385-97.).

Comparison of the combination of MG2-1, MG2-2, MG2-3, MG2-5, and MG2-6 with additional proprietary protein datasets reveals similar Additional protein sequences have been revealed that have the architecture of Motifs commonly found in MG2 family members are presented as SEQ ID NOs: 5631-5638.

Example 2C. - Discovery of the MG3 family of CRISPR systems Analysis of the metagenomic data of Example 1 revealed a new putative CRISPR system: MG3-1 that has not been described so far. The corresponding amino acid sequences of this new enzyme and its exemplary subdomains are presented as SEQ ID NOs: 424, 2245, and 4059. Based on its proximity to other elements of the operon, a putative tracrRNA-containing sequence was identified and included as SEQ ID NO:5498. A detailed domain-level alignment of this sequence pair with Cas9 from Actinomyces naeslundii is shown in FIG.

Comparison of MG3-1 with other unique protein datasets revealed additional protein sequences of similar structure, presented as SEQ NOs: 421-423, 425-431.

Example 2D. -Discovery of MG4, 7, 14, 15, 16, 18, 21, 22, and 23 families using the CRISPR system Analysis of the data from the metagenomic analysis in Example 1 revealed that 9 families with one member each (MG4-5, MG7-2 , MG14-1, MG15-1, MG16-2, MG18-1, MG21-1, MG22-1, MG23-1), a new cluster of previously undescribed putative CRISPR systems has been revealed. Ta. Protein and nucleic acid sequences corresponding to these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 432, 669, 678, 930, 1093, 1354, 1512, 1656, 1756. Putative tracr-containing sequences were identified for each family based on their proximity to other elements of the operon. These sequences are presented in the sequence listing as SEQ ID NOs: 5503-5511, respectively.

Comparing MG4-5, MG7-2, MG14-1, MG15-1, MG16-2, MG18-1, MG21-1, MG22-1, MG23-1 and other proprietary protein datasets, SEQ NO. Additional with similar architecture are presented as: 433-660, 670-677, 679-929, 931-1092, 1094-1353, 1355-1511, 1513-1655, 1657-1755, and 1757-1826. The protein sequence has been revealed. Common motifs for 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: 5687-5693 and SEQ ID NO: 5674-5675 for MG21, SEQ ID NO: 5694-5699 for MG22, and SEQ ID NO: 5700-5717 for MG23. It is presented.

Example 3. -Prophetic--Determination of Protospacer-Adjacent Motif.
The experiment was performed as described by Karvelis et al. Methods. The protospacer-adjacent motif ( PAM) identifies sequence specificity to enable optimal synthetic sequence targeting.

In one example (an in vivo screen), cells with a plasmid encoding any of the enzymes described herein and a guide RNA targeting the protospacer are injected with a plasmid library containing antibiotic resistance genes, and a random The transformed PAM sequence and the flanking protospacer sequence are co-transformed. Plasmids containing functional PAM are cleaved by enzymes, leading to cell death. Deep sequencing of enzyme cleavage-resistant plasmid pools isolated from surviving cells reveals a depleted set of plasmids containing PAMs that allow functional cleavage.

In another example (in vitro screening), a PAM library in the form of DNA plasmids or concatenated repeats can be used to synthesize RNP complexes (e.g., enzymes, tracrRNA and crRNA, or enzymes and hybrid sgRNAs) assembled in vitro or in cell lysates. (including cutting). Free DNA ends resulting from a successful cleavage event are captured by adapter ligation and then subjected to PCR amplification of the product on the PAM side. The amplified library of functional PAMs is subjected to deep sequencing to identify PAMs that license DNA cleavage.

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

Whichever transfection method is chosen, (i) and (ii) are introduced into the cells. An incubation period is allowed to occur 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). Enzyme-mediated cleavage and non-homologous end joining results in the introduction of indels into the genomic DNA in the vicinity of the targeting sequence.

In some embodiments, (i) and (ii) are introduced into the cell along with a third repair nucleotide encoding a region of the genome that is adjacent to the cleavage site of size 25 bp or greater, thereby Oriented repair is promoted. Within these contiguous sequences may be single base pair mutations, functional gene fragments, foreign or native genes for expression, or multiple genes that constitute a biochemical pathway. .

Example 5. -Prophetic--Use of the synthetic CRISPR system described herein in vitro. Any of the enzymes described herein are cloned into an appropriate E. coli expression plasmid containing a purification tag and recombined in E. coli. Expressed and purified using recombinant tags. RNA containing the 5'G followed by 20 nt of targeting and PAM sequences, the tracrRNA binding region of a compatible crRNA, a GAAA linker, and a compatible tracrRNA is synthesized by an appropriate solid-phase RNA synthesis method. Ru. The recombinant enzyme and sgRNA are formulated in an appropriate cleavage buffer containing Mg2+ (e.g. 20mM HEPES pH 7.5, 100mM KCl, 5mM _MgCl2 , 1mM DTT, 5% glycerol) and combined with the targeting sequence. The reaction is initiated by introducing target DNA containing a sequence complementary to the PAM sequence. DNA cleavage is monitored by a suitable assay, such as agarose gel electrophoresis followed by ethidium bromide staining (or a similarly acting DNA intercalating agent) and UV visualization.

Example 6. - (General Protocol) PAM sequence identification/confirmation of the endonucleases described herein PAM sequences were randomly generated that could be cleaved with putative endonucleases expressed in an E. coli lysate-based expression system (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 the tracr sequence under the T7 promoter and a minimal CRISPR array consisting of the T7 promoter followed by repeat-spacer-repeat sequences was transcribed in the same reaction. Successful expression of the endonuclease and tracr sequences in the TXTL system and subsequent CRISPR array processing resulted in an active CRISPR nuclease complex in vitro.

A library of target plasmids containing a spacer sequence matching the minimal array followed by 8N mixed bases (putative PAM sequences) was incubated with the output of the TXTL reaction. The reaction was stopped after 1-3 hours, and DNA was collected using a DNA cleanup kit such as Zymo DCC, AMPure XP beads, QiaQuick, etc. The adapter sequence was blunt-end ligated to DNA with an active PAM sequence that was cleaved by the endonuclease, while uncut DNA was unavailable for ligation. DNA segments containing active PAM sequences were then amplified by PCR using primers specific for the library and adapter sequences. PCR amplification products were separated on a gel and amplicons corresponding to cleavage events were identified. The amplified segment of the cleavage reaction was also used as a template to prepare the NGS library. Sequencing of this resulting library, which was a subset of the original 8N library, revealed the exact PAM-containing sequence of the active CRISPR complex. For PAM tests with a single RNA construct, the same procedure was repeated except that the in vitro transcribed RNA was added along with the plasmid library and the tracr/minimal CRISPR array template was omitted. For the endonucleases for which NGS libraries were prepared, representations of seqLogo (e.g., Huber et al. Nat Methods. 2015 Feb; 12(2):115-21) were constructed and , 32, 33, 34, and 35. The seqLogo module used to construct these representations takes a position-specific weight matrix of a DNA sequence motif (e.g., a PAM sequence) and plots the corresponding sequence logo introduced by Schneider and Stephens. (see, eg, Schneider et al. Nucleic Acids Res. 1990 Oct 25;18(20):6097-100). The characters representing sequences in the seqLogo representation are superimposed on each other for each position of an aligned sequence (eg, a PAM sequence). The height of each letter is proportional to its frequency, and the letters are arranged so that the most frequent letters are on top.

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

Example 8. - (General protocol) In vitro cleavage efficiency of the MG CRISPR complex The endonuclease was expressed as a His-tagged fusion protein from an inducible T7 promoter in a protease-deficient E. coli B strain. Cells expressing His-tagged proteins were lysed by ultrasound, and His-tagged proteins were lysed by Ni-NTA affinity chromatography on a HisTrap FF column (GE Lifescience) on an AKTA Avant FPLC (GE Lifescience). Refined. Eluates were resolved by SDS-PAGE on acrylamide gels (Bio-Rad) and stained with InstantBlue Ultrafast Coomassie (Sigma-Aldrich). Purity was determined using densitometry of protein bands using ImageLab software (Bio-Rad). The purified endonuclease was dialyzed into a storage buffer consisting of 50mM Tris-HCl, 300mM NaCl, 1mM TCEP, 5% glycerol; pH 7.5 and stored at -80°C.

Target DNA containing a spacer sequence and a PAM sequence (eg, determined as in Example 6) was constructed by DNA synthesis. One representative PAM was selected for testing where the PAM has a degenerate base. The target DNA contained a 2200 bp linear DNA obtained from a plasmid by PCR amplification using PAM and a spacer located 700 bp from one end. When the cleavage was successful, fragments of 700 bp and 1500 bp were obtained. Target DNA, in vitro transcribed single RNA, and purified recombinant protein were combined in cleavage buffer (10mM Tris, 100mM NaCl, 10mM _MgCl2 ) containing excess protein and RNA for 5 min. Incubation was for 3 hours, usually 1 hour. The reaction was stopped by addition of RNAse A and incubation for 60 minutes. The reactions are then resolved on a 1.2% TAE agarose gel and the percentage of target DNA cleaved is quantified with ImageLab software.

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 breaks. Therefore, cleavage of genomic DNA can be a fatal event. Taking advantage of this phenomenon, the endonuclease activity was tested in E. coli by recombinantly expressing the endonuclease and tracrRNA in a target strain in which the spacer/target sequence and PAM sequence had been incorporated into the genomic DNA.

In this assay, the PAM sequence is specific for the endonuclease being tested as determined by the method described in Example 6. The sgRNA sequence was determined based on the sequence and predicted structure of tracrRNA. Starting from the 5' end of the repeat, repeat-anti-repeat pairs of 8-12 bp (generally 10 bp) were selected. The 3' ends of the remaining repeats and the 5' ends of tracrRNA were replaced with tetraloops. Generally, the tetraloop was GAAA, but other tetraloops can be used, particularly if the GAAA sequence is predicted to inhibit folding. In such cases, a TTCG tetraloop was used.

A recombinant strain with a PAM sequence incorporated into its genomic DNA was transformed with DNA encoding the endonuclease. The transformants are then chemically adapted to receive 50 ng of a single guide RNA that is specific for the target sequence (“on target”) or non-specific for the target (“non target”). was used for transformation. After heat shock, transformants were collected in SOC at 37°C for 2 hours. Nuclease efficiency was then determined by a 5-fold dilution series incubated in induction medium. Colonies were quantified from a dilution series in triplicate.

Example 10. - (General Protocol) Testing the Genomic Cleavage Activity of the MG CRISPR Complex in Mammalian Cells To demonstrate targeting and cleavage activity in mammalian cells, the MG Cas effector protein sequence was transferred to two mammalian expression vectors: ( Tested a) with an SV40 NLS at the C-terminus and a 2A-GFP tag, and (b) without a GFP tag and with two SV40 NLS sequences, one at the N-terminus and one at the C-terminus. In some instances, the nucleotide sequence encoding the endonuclease is codon-optimized for expression in mammalian cells.

The corresponding single guide RNA sequence (sgRNA) with added targeting sequence is cloned into a second mammalian expression vector. The two plasmids are co-transfected into HEK293T cells. Seventy-two hours after co-transfecting the expression plasmid and sgRNA targeting plasmid into HEK293T cells, DNA is extracted and used for NGS-library construction. To demonstrate the targeting efficiency of the enzyme in mammalian cells, we measure the percent NHEJ through indels 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

Targeted 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. The amplification products were MG1-4 (double guide: gel 1, see lane 3; single guide: gel 6, see lane 2), MG1-5 (gel 2, lane 10), MG1-6 (duplex guide: gel 1, see lane 3), MG1-5 (gel 2, lane 10), Guide: gel 5, see lane 6, single guide: gel 6, see lane 5), MG1-7 (double guide: gel 3, see lane 13, single guide: gel 3, see lane 2) ) (Protein SEQ ID NOs: 1-4, respectively). Sequencing of the PCR products revealed the active PAM sequences of these enzymes, as shown in Table 2.

Synthetic single guide RNAs (sgRNAs) were designed based on the sequence and predicted structure of tracrRNA and are presented as SEQ ID NOs: 5461-5464. The PAM sequence screen of Example 6 was repeated using sgRNA. The results of this experiment are also shown in Table 2 and reveal that the use of sgRNA slightly altered the specificity of PAM.

Targeted endonuclease activity in vitro

The in vitro activity of the MG1-4 endonuclease system (Protein SEQ ID NO: 1 with sgRNA SEQ ID NO: 5461) against target DNA having the 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 length of the spacer/targeting sequence was varied from 18 to 24 nt by replacing Ns in the sequence. The results are shown in FIG. The left panel shows a gel demonstrating DNA cleavage by MG1-4 in combination with the corresponding single guide sgRNAs with different targeting sequence lengths (18-24 nt), and the right panel quantified the same data as a bar graph. show something This data demonstrated that targeting sequences of 18-24 nucleotides were 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 as in Example 9 using the PAM sequence CAGGAAGG. Transformed E. coli were plated in serial dilutions and the results are presented in FIG. 11 (serial dilutions of E. coli are shown in the left panel and quantified growth is shown in the right panel). The significant reduction in growth of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that the genomic DNA was specifically cleaved by the endonuclease in 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. The open reading frames encoding the sequences of MG1-4 (Protein SEQ ID NO: 5527) and MG1-6 (Protein SEQ ID NO: 5529) have a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB). , was cloned into two mammalian expression vectors, one without a GFP tag and with two NLS sequences, 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 the 2-NLS plasmid backbone (MG-16hs). The results of this experiment are shown in FIG. The endonuclease expression vector comprises HEK293T along with a second vector for expressing sgRNA (e.g., SEQ ID NO: 5512 or 5515) having an endonuclease-specific tracr sequence and a guide sequence selected from Tables 3-4. co-transfected into cells. 72 hours after co-transfection, DNA was extracted and used for NGS-library preparation. Cleavage activity was detected by the appearance of internal deletions (NHEJ remnants) in close proximity to the target site sequence. To demonstrate the targeting efficiency of the enzyme in mammalian cells, the proportion of NHEJ through indels in the sequence of the target site was determined and shown in FIG. 12.

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

Targeted endonuclease activity of MG2 family members was confirmed with the myTXTL system as described in Example 6. The results of this assay are shown in Figures 17-20. In the assays shown in Figures 17-20, active proteins that are successfully cleaved from the library give rise to 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 products 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 were plated in serial dilutions and the results are presented in FIG. 34 (serial dilutions of E. coli are shown in the left panel and quantified growth is shown in the right panel). The significant reduction in growth of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by MG1-4 endonuclease in E. coli cells. There is.

Example 13. -Characterization of MG3 family members

PAM specificity, tracrRNA/sgRNA verification

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

Synthetic single guide RNAs (sgRNAs) were designed based on the sequence and predicted structure of tracrRNA and are presented as SEQ ID NOs: 5466-5467. The PAM sequence screen of Example 6 was repeated using sgRNA. The results of this experiment are also shown in Table 6 and reveal that the use of sgRNA slightly altered the specificity of PAM.

Targeted endonuclease activity in vitro

The 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 and the length of the spacer/targeting sequence was varied from 18 to 24 nt by replacing Ns in the sequence. The results are shown in FIG. The upper panel shows a gel demonstrating DNA cleavage by MG3-6 in combination with various sgRNAs with different targeting sequence lengths (18-24 nt), and the lower panel shows the same data quantified as a bar graph. . This data demonstrated that targeting sequences of 18-24 nucleotides were 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 was plated in serial dilutions and the results are presented in FIG. 14 (top panel shows serial dilution of E. coli, bottom panel shows quantified growth). The significantly reduced growth of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that the genomic DNA was being 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 divided into two types: one with a C-terminal SV40 NLS and a 2A-GFP tag (E. coli MG-BB), and one without a GFP tag and with a 1 at the N-terminus. It was cloned into two mammalian expression vectors, one with two NLSs and one at the C-terminus (E. coli pMG5-BB). The endonuclease expression vector was co-transfected into HEK293T cells with a second vector to express the sgRNA described above using a guide sequence selected from Table 7. The results of this experiment are shown in FIG. 72 hours after co-transfection, DNA was extracted and used for NGS-library preparation. Cleavage activity was detected by the appearance of internal deletions (NHEJ remnants) in the vicinity of the target site. The results are shown in FIG.

The target sites encoded on the sgRNA plasmids 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. An amplification product was observed for MG4-2 (double guide: gel 2, lane 9; single guide: gel 10, see lane 7) (SEQ ID NO: 432). Sequencing of the PCR products 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. Amplification products were observed for MG14-1 (double guide: gel 2, 1 lane 4, single guide: gel 3, see lane 8) (SEQ ID NO: 678). Sequencing of the PCR products revealed active PAM sequence specificity as 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 were plated in serial dilutions and the results are presented in FIG. 35 (serial dilutions of E. coli are shown in the left panel and quantified growth is shown in the right panel). The significant reduction in growth of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by MG1-4 endonuclease in E. coli cells. 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. Amplification products were observed for MG15-1 (dual guide: gel 2, lane 7; single guide: gel 3, see lane 9) (SEQ ID NO: 930). Sequencing of the PCR products revealed active PAM sequence specificities 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 length of the spacer/targeting sequence was varied from 18 to 24 nt by replacing Ns in the sequence. The results are shown in FIG. The top panel shows a gel demonstrating DNA cleavage by MG15-1 in combination with various sgRNAs with different targeting sequence lengths (18-24 nt), and the bottom panel shows the same data quantified as a bar graph. . This data demonstrated that targeting sequences of 18-24 nucleotides were 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 GGGTCAA PAM sequence was confirmed using the method described in Example 9. Transformed E. coli were plated in serial dilutions and the results are presented in FIG. 35 (serial dilutions of E. coli are shown in the left panel and quantified growth is shown in the right panel). The significant reduction in growth of E. coli expressing on-target sgRNA compared to E. coli expressing non-target sgRNA indicates that genomic DNA was specifically cleaved by MG1-4 endonuclease in E. coli cells. 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. Amplification products were observed for MG16-2 (see gel 11, lane 17) (SEQ ID NO: 1093). Sequencing of the PCR products revealed active PAM sequence specificities 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. An amplification product was observed for MG18-1 (double guide: gel 2, lane 9; single guide: gel 11, see lane 12) (SEQ ID NO: 1354). Sequencing of the PCR products revealed active PAM sequence specificities 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. An amplification product was observed for MG21-1 (see gel 11, lane 2) (SEQ ID NO: 1512). Sequencing of the PCR products revealed active PAM sequence specificities 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. In the assays shown in Figures 17-20, active proteins that are successfully cleaved from the library give rise to a band of approximately 170 bp in the gel. An amplification product was observed for MG22-1 (see gel 11, lane 3) (Protein SEQ ID NO: 1656). Sequencing of the PCR products revealed active PAM sequence specificities 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 results in a product that migrates at approximately 170 bp in the gel, as shown in Figures 17-20. An amplification product was observed for MG23-1 (see gel 11, lane 4) (SEQ ID NO: 1756). Sequencing of the PCR products revealed active PAM sequence specificity for these enzymes, detailed in Table 15 below.

The systems of the present disclosure can be used in a variety of applications, such as, for example, nucleic acid editing (eg, gene editing), binding to nucleic acid molecules (eg, sequence-specific binding), and the like. Such systems can, for example, inject high-value small molecules, macromolecules, or secondary metabolites to inactivate the virus by targeting the viral genome or rendering it incapable of infecting host cells. by exogenous small molecules and nucleotides as biosensors, to establish gene driving elements for evolutionary selection, to add genes or change metabolic pathways to manipulate organisms to produce Disease-causing genes, such as inactivating enzymes combined with probes to target and detect specific nucleotide sequences (e.g., sequences encoding antibiotic resistance in bacteria) to detect cellular perturbations. as a diagnostic tool for detecting elements that are likely to cause disease in a subject (e.g., through cleavage of reverse transcribed viral RNA or amplified DNA sequences encoding disease-causing mutations). It may be used to address (e.g. remove or replace) genetically inherited mutations to inactivate the gene and thereby confirm the function of the gene within the cell.

While preferred embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. Although the invention has been described with respect to the foregoing specification, the description and illustrations of embodiments herein are not intended to be construed in a limiting sense. Many modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it will be understood that all aspects of the invention are not limited to the particular depictions, configurations, or relative proportions described herein, depending on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. Therefore, it is contemplated that the invention extends to any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (170)

An engineered nuclease system, the engineered nuclease system comprising:
(a) An endonuclease comprising a RuvC_III domain and a HNH domain, wherein the endonuclease is derived from a difficult-to-cultivate microorganism, and wherein the endonuclease is a class 2 type II Cas endonuclease. , endonuclease, and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease, said engineered guide ribonucleic acid structure comprising:
(i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence;
(ii) an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease;
Engineered nuclease system. 2. 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. engineered nuclease system. An engineered nuclease system, the engineered nuclease system comprising:
(a) an endonuclease comprising 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 said endonuclease, said engineered guide ribonucleic acid structure comprising:
(i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence;
(ii) an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease;
Engineered nuclease system. An engineered nuclease system, the engineered nuclease system comprising:
(a) an endonuclease configured to bind to a protospacer adjacent motif (PAM) sequence comprising SEQ ID NO: 5512-5537, wherein the endonuclease is a class 2 type II Cas endonuclease; , endonuclease, and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease, said engineered guide ribonucleic acid structure comprising:
(i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence;
(ii) an engineered guide ribonucleic acid structure comprising a tracr ribonucleic acid sequence configured to bind to the endonuclease;
Engineered nuclease system. 5. The engineered nuclease system of claim 4, wherein the endonuclease is derived from a refractory microorganism. Engineered nuclease system according to any one of claims 4-5, wherein the endonuclease is not engineered to bind to different PAM sequences. The endonucleases include Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, Cas13c endonuclease. or Cas13d endonuclease 5. The engineered nuclease system of claim 4, wherein the engineered nuclease system is free. 5. The engineered nuclease system of claim 4, wherein the endonuclease has less than 80% identity to Cas9 endonuclease. Engineered nuclease system according to 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 to 90 contiguous nucleotides selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. , an engineered nuclease system according to any one of claims 1-9. An engineered nuclease system, the engineered nuclease system comprising:
(a) an engineered guide ribonucleic acid structure, the engineered guide ribonucleic acid structure comprising:
(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 said tracr ribonucleic acid sequence is selected from any one of SEQ ID NO: 5476-5511 and SEQ ID NO: 5538. a tracr ribonucleic acid sequence comprising a sequence having at least 80% sequence identity over about 60 to 90 contiguous nucleotides;
engineered guide ribonucleic acid structure;
(b) a class 2 type II Cas endonuclease configured to bind to the engineered guide ribonucleic acid;
Engineered nuclease system. The engineered endonuclease of any of claims 1-3 or 11, wherein the endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence selected from the group comprising SEQ ID NOs: 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. 12. The engineered nuclease system of any one of claims 1-11, wherein the engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising the guide ribonucleic acid sequence and the tracr ribonucleic acid sequence. 15. The operation of 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. Engineered nuclease system according to any one of claims 1-15, wherein the guide ribonucleic acid sequence is 15-24 nucleotides long. The engineered endonuclease of any one of claims 1-16, wherein the endonuclease comprises one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. Nuclease system. Engineered nuclease system according to any one of claims 1-17, wherein the NLS comprises a sequence selected from SEQ ID NOs: 5597-5612. 5' to 3', a first homology arm comprising a sequence of at least 20 nucleotides 5' of said target deoxyribonucleic acid sequence; a synthetic DNA sequence of at least 10 nucleotides; and 3' of said target deoxyribonucleic acid sequence; and a second homology arm comprising a sequence of at least 20 nucleotides. Nuclease system. 20. 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. 21. The engineered nuclease system of any one of claims 1-20, wherein the engineered nuclease system further comprises a source of Mg2 ⁺ . 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. Engineered nuclease system according to any one of claims 1-22, wherein the endonuclease is derived from a bacterium belonging to the genus Dermabacter. Engineered nuclease system according to any one of claims 1-22, wherein the endonuclease is derived from a bacterium belonging to the phylum Verrucomicrobia, Candidatus Peregrinibacteria, or Candidatus Melainabacteria. 23. The endonuclease is derived from a bacterium comprising a 16S rRNA gene with at least 90% identity to any one of SEQ ID NOs: 5592-5595. Engineered nuclease system. The engineered HNH domain of any one of claims 1-25, wherein the HNH domain comprises a sequence with at least 70% or at least 80% identity to any one of SEQ ID NOs: 5638-5460. nuclease system. 27. 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 thereto. 1-, 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: 1827-1830 or SEQ ID NO: 1827-2140. 28. The engineered nuclease system according to any one of 27. Claims 1-28, wherein 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: 3638-3641 or SEQ ID NO: 3638-3954. An engineered nuclease system according to any one of . 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 NOs: 5615-5632. The engineered nuclease system according to any one of the above. Claim 1-, 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: 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. 32. An engineered nuclease system according to any one of claims 1-31, comprising a sequence. The guide RNA structure includes an RNA sequence predicted to include a hairpin consisting of a stem and a loop, where the stem is at least 10, at least 12, or at least 14 base pairs of ribonucleotides, and the loop is 33. Engineered nuclease system according to any one of claims 1-32, comprising an asymmetric bulge of up to 4 base pairs. 34. 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. engineered 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) An engineered nuclease system according to 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 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 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: 5463 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: 5529. 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. Claim 1-, 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: 2141-2142 or SEQ ID NO: 2141-2241. 28. The engineered nuclease system according to any one of 27. Claim 1-, 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: 3955-3956 or SEQ ID NO: 3955-4055. The engineered nuclease system according to any one of 27 or 39. 39. Claims 1-27 or 39, 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: 5632-5638. -40. The engineered nuclease system according to any one of -40. Claim 1-, 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: 320-321 or SEQ ID NO: 320-420. 27 or 39-41. The guide RNA structure is a sequence that 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. 43. An engineered nuclease system according to any one of claims 1-27 or 39-42, comprising: 44. The guide RNA structure comprises a tracr ribonucleic acid sequence, comprising a hairpin comprising at least 8, at least 10, or at least 12 base pairs of ribonucleotides. engineered nuclease system. 45. According to any one of claims 1-27 or 39-44, 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. engineered nuclease system. a) the endonuclease comprises 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 comprises 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. 28. The endonuclease according to any one of claims 1-27, 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 NOs: 2245-2246. The engineered nuclease system described. 49. Any 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 NOs: 4059-4060. The engineered nuclease system described in. 48. Claims 1-27 or 48, 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 NOs: 5639-5648. -49. The engineered nuclease system according to any one of -49. Any 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 NOs: 424-425. The engineered nuclease system according to any one of or claim 1-27, wherein 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. 48-52. The engineered nuclease system according to any one of 48-51. The guide RNA structure comprises a guide ribonucleic acid sequence predicted to include a hairpin with an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence, wherein , wherein the tracr ribonucleic acid sequence comprises, 5' to 3', a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin. Engineered nuclease system according to any one of paragraphs 1-27 or 48-52. Claim 1-, 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: 2242-2244 or SEQ ID NO: 2247-2249. 28. The engineered nuclease system according to any one of 27. Claim 1-, 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: 4056-4058 and SEQ ID NO: 4061-4063. 55. The engineered nuclease system according to any one of 27 or 54. 54. Claims 1-27 or 54, 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 NOs: 5639-5648. -55. The engineered nuclease system according to any one of -55. Claim 1-, 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: 421-423 or SEQ ID NO: 426-428. 27 or 54-56. The guide RNA structure has at least 70%, 80%, or 90% identical sequences. The guide RNA structure comprises a guide ribonucleic acid sequence predicted to include a hairpin with an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence, wherein , wherein the tracr ribonucleic acid sequence comprises, 5' to 3', a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin. An engineered nuclease system according to any one of paragraphs 1-27 or 54-58. Any 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 NOs: 5517-5518 or SEQ ID NOs: 5532-5534. An engineered nuclease system according to one. a) the endonuclease comprises 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 comprises 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 comprises 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. 28. The endonuclease of claims 1-27, 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: 2253 or SEQ ID NO: 2253-2481. An engineered nuclease system according to any one. or claim 1-27, 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: 4067 or SEQ ID NO: 4067-4295. 64. The engineered nuclease system according to any one of 64. 66. The engineered nuclease system of any one of claims 1-27 or 64-65, wherein the endonuclease comprises the peptide motif of SEQ ID NO: 5649. or claim 1-27, 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: 432 or SEQ ID NO: 432-660. The engineered nuclease system according to any one of 64-66. 64. Claims 1-27 or 64, 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: 5468 or SEQ ID NO: 5503. -67. The engineered nuclease system according to any one of -67. 69. The engineered nuclease of 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 comprises 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. 28. The endonuclease according to any one of claims 1-27, 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 NOs: 2482-2489. The engineered nuclease system described. 72. Any 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 NOs: 4296-4303. The engineered nuclease system described in. Any 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 NOs: 661-668. The engineered nuclease system according to any one of the above. 28. The endonuclease according to any one of claims 1-27, 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 NOs: 2490-2498. The engineered nuclease system described. 75. Any 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 NOs: 4304-4312. The engineered nuclease system described in. Any 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 NOs: 669-677. The engineered nuclease system according to any one of the above. Any 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. An engineered nuclease system according to one. 28. The endonuclease of claims 1-27, 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: 2499 or SEQ ID NO: 2499-2750. An engineered nuclease system according to any one. or claim 1-27, 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: 4313 or SEQ ID NO: 4313-4564. 78. The engineered nuclease system according to any one of 78. 78. Claims 1-27 or 78, 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 NOs: 5650-5667. -79. or claim 1-27, 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: 678 or SEQ ID NO: 678-929. 78-80. Any 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. 83. 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 comprises 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. . 28. The endonuclease of claims 1-27, 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: 2751 or SEQ ID NO: 2751-2913. An engineered nuclease system according to any one. 27 or 27, 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: 4565 or SEQ ID NO: 4565-4727. 85. The engineered nuclease system according to any one of 85. 85. Claims 1-27 or 85, 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 NOs: 5668-5678. -86. The engineered nuclease system according to any one of -86. or claim 1-27, 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: 930 or SEQ ID NO: 930-1092. 85-87. Any 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. 89. According to any one of claims 1-27 or 85-89, 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. engineered nuclease system. a) the endonuclease comprises 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. . 28. The endonuclease of claims 1-27, 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: 2914 or SEQ ID NO: 2914-3174. An engineered nuclease system according to any one. or claim 1-27, 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: 4728 or SEQ ID NO: 4728-4988. 92. The engineered nuclease system according to any one of 92. According to any one of claims 1-27 or 92-93, 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. The engineered nuclease system described. 27 or 27, 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: 1093 or SEQ ID NO: 1093-1353. 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. , an engineered nuclease system according to any one of claims 1-27 or 92-95. 97. The guide RNA structure comprises a tracr ribonucleic acid sequence predicted to include at least two hairpins comprising ribonucleotides of less than 5 base pairs. engineered nuclease system. 98. 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 comprises 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. 28. The endonuclease of claims 1-27, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 3175 or a sequence selected from the group consisting of SEQ ID NO: 3175-3330. An engineered nuclease system according to any one. 27 or 27, 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: 4989 or SEQ ID NO: 4989-5146. 100. The engineered nuclease system according to any one of 100. 100. Claims 1-27 or 100, 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 NOs: 5679-5686. -101. The engineered nuclease system according to any one of -101. or claim 1-27, 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: 1354 or SEQ ID NO: 1354-1511. 100-102. The engineered nuclease system according to any one of 100-102. 100. Claim 1-27 or 100, 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: 5472 or SEQ ID NO: 5508. -103. The engineered nuclease system according to any one of -103. 105. 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. engineered nuclease system. a) the endonuclease comprises 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. . 28. The endonuclease of claims 1-27, 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: 3331 or SEQ ID NO: 3331-3474. An engineered nuclease system according to any one. or claim 1-27, 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: 5147 or SEQ ID NO: 5147-5290. 107. 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. An engineered nuclease system according to any one of claims 1-27 or 107-108. or claim 1-27, 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: 1512 or SEQ ID NO: 1512-1655. 107-109. The engineered nuclease system according to any one of 107-109. 107. Claims 1-27 or 107, 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: 5473 or SEQ ID NO: 5509. -110. 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 comprises 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. 28. The endonuclease of claims 1-27, 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: 3475 or SEQ ID NO: 3475-3568. An engineered nuclease system according to any one. or claim 1-27, 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: 5291 or SEQ ID NO: 5291-5389. 114. The engineered nuclease system according to any one of 114. 114. Claims 1-27 or 114, 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 NOs: 5694-5699. -115. or claim 1-27, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1656 or a sequence selected from the group consisting of SEQ ID NO: 1656-1755. 114-116. The engineered nuclease system according to any one of 114-116. Any 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. 119. 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 comprises 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. 28. The endonuclease of claims 1-27, 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: 3569 or SEQ ID NO: 3569-3637. An engineered nuclease system according to any one. or claim 1-27, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 5390 or a sequence selected from the group consisting of SEQ ID NO: 5390-5460. 122. The engineered nuclease system according to any one of 121. 1-27 or 121-, 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: 5700-5717. 122. The engineered nuclease system according to any one of 122. or claim 1-27, wherein the endonuclease comprises a sequence that is at least 70%, 80%, or 90% identical to SEQ ID NO: 1756 or a sequence selected from the group consisting of SEQ ID NO: 1756-1826. 121-123. Any 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. 126. 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 comprises 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. 128. The engineered nuclease system of any one of claims 1-127, wherein sequence identity is determined by a BLASTP, CLUSTALW, MUSCLE, MAFFT, or Smith-Waterman homology search algorithm. The sequence identity was determined using the BLOSUM62 scoring matrix with parameters of wordlength (W) of 3, expectation (E) of 10, and gap costs of 11 existence, 1 extension, and conditional. 129. The engineered nuclease system of claim 128, as determined by a BLASTP homology search algorithm using a compositional score matrix adjustment. An engineered guide ribonucleic acid polynucleotide, the engineered guide ribonucleic acid polynucleotide comprising:
a) a DNA targeting segment comprising a nucleotide sequence complementary to a target sequence in a target DNA molecule;
b) a protein binding segment comprising two complementary stretches of nucleotides that hybridize to form a double-stranded RNA (dsRNA) duplex;
wherein said two complementary stretches of nucleotides are covalently linked to each other at an intervening nucleotide, and
wherein said engineered guide ribonucleic acid polynucleotide forms a complex with an endonuclease comprising a RuvC_III domain having at least 75% sequence identity to any one of SEQ ID NOs: 1827-3637; configured to target the complex to a target sequence of a target DNA molecule;
Engineered guide ribonucleic acid polynucleotides. 131. The engineered guide ribonucleic acid polynucleotide of claim 130, wherein said DNA targeting segment is located 5' of both of said two complementary stretches of nucleotides. a) the protein binding segment has 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) said protein binding segment is at least 70%, at least 80%, or at least 90% 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. comprising a sequence having an 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 NOs: 5498-5499;
d) the protein binding segment has 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) the protein binding segment comprises a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5506;
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 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 a sequence having at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 5510, or
m) the engineered protein binding segment of 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. Guided ribonucleic acid polynucleotide. a) said guide ribonucleic acid polynucleotide comprises an RNA sequence comprising a hairpin comprising a stem and a loop, wherein said stem comprises at least 10, at least 12, or at least 14 base pairs of ribonucleotides; contains an asymmetric bulge within four base pairs,
b) the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence predicted to include a hairpin comprising at least 8, at least 10, or at least 12 base pairs of ribonucleotides;
c) said guide ribonucleic acid polynucleotide comprises a guide ribonucleic acid sequence predicted to include a hairpin having an uninterrupted base-paired region comprising at least eight nucleotides of the guide ribonucleic acid sequence and at least eight nucleotides of the tracr ribonucleic acid sequence; tracr ribonucleic acid sequence comprises, 5' to 3', a first hairpin and a second hairpin, wherein the first hairpin has a longer stem than the second hairpin. has, or
d) the engineered ribonucleic acid polynucleotide of any of claims 130-132, wherein the guide ribonucleic acid polynucleotide comprises a tracr ribonucleic acid sequence predicted to include at least two hairpins comprising ribonucleotides of less than 5 base pairs. guided ribonucleic acid polynucleotide. A deoxyribonucleic acid polynucleotide encoding an 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 the nucleic acid encodes a class 2 type II Cas endonuclease comprising a RuvC_III domain and a HNH domain; Here, the class 2 type II Cas endonuclease is a nucleic acid derived from a difficult-to-cultivate microorganism. A nucleic acid comprising an engineered nucleic acid sequence optimized for expression in an organism, wherein said nucleic acid has at least 70% sequence identity to any one of SEQ ID NOs: 1827-3637. 1. A nucleic acid encoding an endonuclease comprising a RuvC_III domain with a specific property. 137. The endonuclease according to any one of claims 135-136, wherein 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. Nucleic acid. 138. The nucleic acid of any one of claims 135-137, wherein the endonuclease comprises SEQ ID NO: 5572-5591 or a variant thereof having at least 70% sequence identity thereto. 139. According to any one of claims 135-138, the endonuclease comprises a sequence encoding one or more nuclear localization sequences (NLS) proximal to the N-terminus or C-terminus of the endonuclease. of nucleic acids. 140. The nucleic acid of claim 139, wherein the NLS comprises a sequence selected from SEQ ID NOs: 5597-5612. 141. The nucleic acid according to any one of claims 135-140, wherein the organism is a prokaryote, bacterium, eukaryote, fungus, plant, mammal, rodent, or 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 NOs: 5572-5575;
b) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 5576-5577;
c) the nucleic acid sequence has at least 70%, 80%, or 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 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 a 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 comprising a nucleic acid sequence encoding a class 2 type II Cas endonuclease comprising a RuvC_III domain and an HNH domain, wherein the class 2 type II Cas endonuclease is derived from a difficult-to-cultivate microorganism. A vector comprising the nucleic acid according to any of claims 135-143. further comprising a nucleic acid encoding an engineered guide ribonucleic acid structure configured to form a complex with said class 2 type II Cas endonuclease, said engineered guide ribonucleic acid structure comprising:
a) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence;
b) a tracr ribonucleic acid sequence configured to bind to said class 2 type II Cas endonuclease;
146. The vector of any of claims 144-145, comprising: 147. The vector of any of claims 144-146, wherein the vector is a plasmid, a minicircle, a CELiD, a virion derived from an adeno-associated virus (AAV), or a lentivirus. A cell comprising a vector according to any of claims 144-147. 147. A method of producing an endonuclease, the method comprising culturing the cell of claim 146. A method for linking, cleaving, labeling, or modifying double-stranded deoxyribonucleic acid polynucleotides, the method comprising:
(a) a class 2 type II Cas endonuclease in complex with a class 2 type II Cas endonuclease and an engineered guide ribonucleic acid structure configured to bind to the double-stranded deoxyribonucleic acid polynucleotide; contacting the double-stranded deoxyribonucleic acid polynucleotide,
(b) the double-stranded deoxyribonucleic acid polynucleotide comprises a protospacer adjacent motif (PAM), and
(c) the PAM includes a sequence selected from the group consisting of SEQ ID NO: 5512-5526 or SEQ ID NO: 5527-5537;
Method. 150. 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 a PAM. . 152. 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 include Cas9 endonuclease, Cas14 endonuclease, Cas12a endonuclease, Cas12b endonuclease, Cas12c endonuclease, Cas12d endonuclease, Cas12e endonuclease, Cas13a endonuclease, Cas13b endonuclease, C as13c endonuclease , or Cas13d endonuclease. 154. The method of any one of claims 149-153, wherein the class 2 type II Cas endonuclease is derived from a refractory microorganism. 155. The double-stranded deoxyribonucleic acid polynucleotide is a eukaryotic, plant, fungal, mammalian, rodent, or human double-stranded deoxyribonucleic acid polynucleotide according to any one of claims 149-154. the method of. a) said PAM comprises a sequence selected from the group consisting of SEQ ID NOs: 5512-5515 and SEQ ID NOs: 5527-5530;
b) the PAM comprises SEQ ID NO: 5516 or SEQ ID NO: 5531;
c) said PAM comprises SEQ ID NO: 5539;
d) the PAM comprises SEQ ID NO: 5517 or SEQ ID NO: 5518;
e) said 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) said 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 SEQ ID NO: 5525, or
l) the PAM comprises SEQ ID NO: 5526;
156. A method according to any one of claims 149-155. 130. A method of modifying a target nucleic acid locus, the method comprising the step of delivering any of the engineered nuclease systems of any one of claims 1-129 to the target nucleic acid locus. , wherein the endonuclease is configured to form a complex with the engineered guide ribonucleic acid structure, wherein the complex is configured to form a complex with the engineered guide ribonucleic acid structure, wherein upon binding of the complex to the target nucleic acid locus, the endonuclease A method, wherein the complex is configured to modify said target nucleic acid locus. 157. The method of claim 156, wherein modifying the target nucleic acid locus comprises binding, nicking, cleaving, or labeling the target nucleic acid locus. 159. The method of any of claims 156-158, wherein the target nucleic acid locus comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). 160. The method of claim 159, wherein the target nucleic acid comprises genomic DNA, viral DNA, viral RNA, or bacterial DNA. 161. The method of any one of claims 156-160, wherein the target nucleic acid locus is in vitro. 161. The method of any one of claims 156-160, wherein the target nucleic acid locus is intracellular. 163. 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. 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; 164. A method according to any one of claims 162-163. 164. The method of claim 162-163, wherein delivering the engineered nuclease system to the target nucleic acid locus comprises delivering a nucleic acid comprising an open reading frame encoding the endonuclease. Method. 165. The method of claim 164, wherein the nucleic acid comprises a promoter to which the open reading frame encoding the endonuclease is operably linked. 164. Any one of claims 162-163, wherein delivering the engineered nuclease system to the target nucleic acid locus comprises delivering capped mRNA comprising the open reading frame encoding the endonuclease. The method described in. 164. The method of any one of claims 162-163, wherein delivering the engineered nuclease system to the target nucleic acid locus comprises delivering translated polypeptide. 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 a ribonucleic acid (RNA) pol III promoter. 164. A method according to any one of claims 162-163, comprising: 170. The method of any one of claims 156-169, wherein the endonuclease causes a single-strand break or a double-strand break at or proximal to the target nucleic acid locus.