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 composition comprising:
(a) an endonuclease comprising a RuvC_III domain and an HNH domain, wherein said endonuclease comprises a sequence having at least 90% sequence identity to SEQ ID NO: 421; 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 (DNA) sequence; and
ii) a tracr ribonucleic acid sequence configured to form a complex with said endonuclease.
2. An isolated cell comprising said engineered composition of claim 1 .
3. The isolated cell of claim 2 , further comprising
c) a target DNA sequence comprising a protospacer adjacent motif sequence comprising 5′-RGG-3′, wherein R denotes A or G.
4. The engineered composition 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 of SEQ ID NO: 5495.
5. The engineered composition of claim 1 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence of SEQ ID NO: 5517.
6. The engineered composition of claim 1 , wherein said engineered guide ribonucleic acid structure comprises at least two ribonucleic acid polynucleotides.
7. The engineered composition 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.
8. The engineered composition of claim 1 , wherein said guide ribonucleic acid structure is complementary to a prokaryotic, bacterial, archaeal, eukaryotic, fungal, plant, mammalian, or human genomic sequence.
9. The engineered composition of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N- or C-terminus of said endonuclease.
10. The engineered composition of claim 9 , wherein said one or more NLSs comprises a sequence selected from any one of SEQ ID NOs: 5597-5612.
11. The engineered composition 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 DNA 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 DNA sequence.
12. The engineered composition of claim 1 , wherein said engineered composition further comprises a source of Mg 2+ .
13. The engineered composition of claim 4 , wherein said endonuclease and said tracr ribonucleic acid sequence are derived from distinct bacterial species within a same phylum.
14. The engineered composition of claim 1 , wherein said engineered guide ribonucleic acid structure comprises an RNA sequence predicted to comprise a hairpin consisting of a stem and a loop, wherein said stem comprises at least 10 base-paired ribonucleotides and an asymmetric bulge within 4 base pairs of said loop.
15. The engineered composition of claim 4 , wherein said tracr ribonucleic acid sequence of said engineered guide ribonucleic acid structure comprises a hairpin comprising at least 8 base-paired ribonucleotides.
16. The engineered composition 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 said guide ribonucleic acid sequence and at least 8 nucleotides of said 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.