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 . An engineered nuclease system, comprising:
(a) an endonuclease configured to have selectivity for a protospacer adjacent motif sequence of nnGGG (SEQ ID NO: 5517);
wherein said endonuclease comprises a RuvC_III domain,
wherein said RuvC_III domain comprises a sequence having at least 80% identity to SEQ ID NO: 2242, or a variant thereof,
wherein said endonuclease is a class 2, type II Cas endonuclease; and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease comprising:
(i) a guide ribonucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and
(ii) a tracr ribonucleic acid sequence configured to bind to said endonuclease.
2 . (canceled)
3 . (canceled)
4 . The engineered nuclease system of claim 1 , wherein said endonuclease further comprises an HNH domain.
5 . The engineered nuclease system of claim 1 , wherein said tracr ribonucleic acid sequence comprises a sequence with at least 80% sequence identity to about 60 to 90 consecutive nucleotides selected from any one of SEQ ID NOs: 5476-5511 and SEQ ID NO: 5538.
6 . (canceled)
7 . The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides.
8 . The engineered nuclease system 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.
9 . The engineered nuclease system of claim 1 , wherein said guide ribonucleic acid sequence is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.
10 . The engineered nuclease system of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
11 . The engineered nuclease system of claim 10 , wherein said one or more NLSs comprises a sequence selected from SEQ ID NOs: 5597-5612.
12 . The engineered nuclease system of claim 1 , further comprising a single- or double-stranded DNA repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said target deoxyribonucleic acid sequence.
13 . The engineered nuclease system of claim 1 , wherein said engineered nuclease system further comprises a source of Mg2+.
14 . The engineered nuclease system of claim 1 , wherein said endonuclease and said tracr ribonucleic acid sequence are derived from distinct bacterial species within a same phylum.
15 . The engineered nuclease system of claim 1 , wherein said endonuclease comprises SEQ ID NOs: 421 or a variant thereof having at least 55% identity thereto.
16 . The engineered nuclease system of claim 1 , wherein said guide RNA structure comprises an RNA sequence predicted to comprise a hairpin consisting of a stem and a loop, wherein the stem comprises at least 10base-paired ribonucleotides, and an asymmetric bulge within 4 base pairs of the loop.
17 . The engineered nuclease system of claim 1 , wherein said tracr ribonucleic acid sequence of said engineered guide ribonucleic acid structure comprises a hairpin comprising at least 8base-paired ribonucleotides.
18 . The engineered nuclease system of claim 1 , wherein said guide ribonucleic acid sequence of said engineered guide ribonucleic acid structure is predicted to comprise a hairpin with an uninterrupted base-paired region comprising at least 8 nucleotides of a guide ribonucleic acid sequence and at least 8 nucleotides of a tracr ribonucleic acid sequence, and wherein said tracr ribonucleic acid sequence comprises, from 5′ to 3′, a first hairpin and a second hairpin, wherein said first hairpin has a longer stem than said second hairpin.
19 . (canceled)
20 . (canceled)
21 . (canceled)
22 . (canceled)
23 . The engineered nuclease system of claim 1 , further comprising a target DNA sequence comprising the sequence 5′-nnGGG-3′ which is 3′ to said target deoxyribonucleic acid sequence.