Enzymes with RuvC domains
The present disclosure provides for endonuclease enzymes having distinguishing domain features, as well as methods of using such enzymes or variants thereof.
1 . A method of modifying a target nucleic acid in an isolated cell, said method comprising introducing to said cell:
(a) an endonuclease or a nucleic acid encoding said endonuclease, wherein said endonuclease comprises a RuvC_III domain and an HNH domain, wherein said endonuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 421; and
(b) an engineered guide ribonucleic acid structure or a nucleic acid encoding said engineered ribonucleic acid structure, wherein said engineered guide ribonucleic acid structure comprises:
i) a guide ribonucleic acid sequence configured to hybridize to a portion of said target nucleic acid; and
ii) a tracr ribonucleic acid sequence configured to form a complex with said endonuclease.
2 . The method of claim 1 , wherein said endonuclease comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 421.
3 . The method of claim 2 , wherein said endonuclease comprises the amino acid sequence of SEQ ID NO: 421.
4 . The method of claim 1 , wherein said RuvC_III domain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2242.
5 . The method of claim 4 , wherein said RuvC_III domain comprises the sequence of SEQ ID NO: 2242.
6 . The method of claim 1 , wherein said HNH domain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 4056.
7 . The method of claim 6 , wherein said HNH domain comprises the sequence of SEQ ID NO: 4056.
8 . The method of claim 1 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence of SEQ ID NO: 5517.
9 . The method of claim 1 , wherein said endonuclease is a class 2, type II Cas endonuclease.
10 . The method of claim 1 , wherein said tracr ribonucleic acid sequence comprises a sequence having at least 80% sequence identity to about 60 to 90 consecutive nucleotides of SEQ ID NO: 5495.
11 . The method of claim 1 , wherein said endonuclease and said tracr ribonucleic acid sequence are derived from distinct bacterial species within a same phylum.
12 . The method of claim 1 , wherein said engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides.
13 . The method of claim 1 , wherein said engineered guide ribonucleic acid structure comprises one ribonucleic acid polynucleotide comprising said guide ribonucleic acid sequence and said tracr ribonucleic acid sequence.
14 . The method of claim 1 , wherein said guide ribonucleic acid sequence is complementary to a eukaryotic, mammalian, or human genomic sequence.
15 . The method of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
16 . The method of claim 15 , wherein said one or more NLSs comprises a sequence selected from any one of SEQ ID NOs: 5597-5612.
17 . The method of claim 1 , further comprising introducing into said cell a single-stranded or double-stranded deoxyribonucleic repair template comprising from 5′ to 3′: a first homology arm comprising a sequence 5′ to said target nucleic acid sequence, a synthetic deoxyribonucleic acid sequence, and a second homology arm comprising a sequence 3′ to said target nucleic acid sequence.
18 . The method of claim 1 , wherein said modifying comprises binding, nicking, cleaving, or marking said target nucleic acid.
19 . The method of claim 18 , wherein said target nucleic acid comprises genomic deoxyribonucleic acid (DNA).
20 . The method of claim 1 , wherein said cell is a eukaryotic cell, an animal cell, a mammalian cell, a primate cell, or a human cell.