Class II, type II CRISPR systems
The present disclosure provides for endonuclease enzymes as well as methods of using such enzymes or variants thereof.
1. An engineered nuclease system comprising:
(a) an endonuclease comprising a RuvC-I domain and an HNH domain; and
(b) an engineered guide ribonucleic acid (RNA) structure capable of forming a complex with said endonuclease comprising:
(i) a guide RNA sequence capable of hybridizing to a target deoxyribonucleic acid (DNA) sequence; and
(ii) an RNA sequence capable of binding to said endonuclease, wherein said RNA sequence comprises a sequence with at least 90% sequence identity to nonvariable nucleotides of any one of SEQ ID NOs: 203, 202, 613, or 614,
wherein said endonuclease comprises a sequence with at least 70% sequence identity to SEQ ID NO: 2.
2. The engineered nuclease system of claim 1 , wherein said endonuclease is an archaeal endonuclease.
3. The engineered nuclease system of claim 1 , wherein said endonuclease is a class 2, type II Cas endonuclease.
4. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises an arginine-rich region comprising an RRxRR motif or a domain with PF14239 homology.
5. The engineered nuclease system of claim 4 , wherein said arginine rich region or said domain with PF 14239 homology has at least 85% identity to an arginine rich region or a domain with PF14239 homology of any one of SEQ ID NOs: 1-198, 221-459, 463-612, or 617-668.
6. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises a recognition (REC) domain.
7. The engineered nuclease system of claim 6 , wherein said REC domain has at least 85% identity to a REC domain of any one of SEQ ID NOs: 1-198, 221-459, 463-612, or 617-668.
8. The engineered nuclease system of claim 1 , wherein said endonuclease further comprises a bridge helix (BH) domain, a wedge (WED) domain, and a PAM-interacting (PI) domain.
9. The engineered nuclease system of claim 8 , wherein said BH domain, said WED domain, or said PI domain has at least 85% identity to a BH domain, a WED domain, or a PI domain of any one of SEQ ID NOs: 1-198, 221-459, 463-612, or 617-668.
10. The engineered nuclease system of claim 1 , wherein said endonuclease has less than 80% identity to a SpCas9 endonuclease.
11. The engineered nuclease system of claim 1 , wherein said sequence identity is determined by a BLASTP homology search algorithm using parameters of a wordlength (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix setting gap costs at existence of 11, extension of 1, and using a conditional compositional score matrix adjustment.
12. The engineered nuclease system of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to an N-terminus of said endonuclease.
13. 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 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.
14. The engineered nuclease system of claim 13 , wherein said first homology arm or said second homology arm comprises a sequence of at least 40 nucleotides.
15. The engineered nuclease system of claim 1 , wherein said endonuclease comprises a sequence with at least about 85% sequence identity to SEQ ID NO: 2.
16. The engineered nuclease system of claim 1 , wherein said endonuclease has a molecular weight of about 120 kDa or less, 100 kDa or less, 90 kDa or less, or 60 kDa or less.
17. The engineered nuclease system of claim 1 , wherein said engineered guide RNA structure comprises a single RNA polynucleotide comprising said guide RNA sequence and said RNA sequence capable of binding to said endonuclease.
18. The engineered nuclease system of claim 1 , wherein said guide RNA sequence is complementary to a eukaryotic, fungal, plant, mammalian, or human genomic sequence.
19. The engineered nuclease system of claim 1 , wherein said guide RNA sequence is 15-24 nucleotides in length.
20. The engineered nuclease system of claim 1 , wherein said endonuclease comprises one or more nuclear localization sequences (NLSs) proximal to a C-terminus of said endonuclease.
21. The engineered nuclease system of claim 1 , wherein said engineered guide RNA structure comprises an RNA sequence predicted to comprise a hairpin comprising a stem and a loop, wherein said stem comprises at least 12 pairs of ribonucleotides.
22. The engineered nuclease system of claim 21 , wherein said engineered guide RNA structure further comprises a second stem and a second loop, wherein said second stem comprises at least 5 pairs of ribonucleotides.
23. The engineered nuclease system of claim 21 , wherein said engineered guide RNA structure further comprises an RNA structure comprising at least two hairpins.
24. The engineered endonuclease system of claim 1 , wherein said RNA sequence comprises a sequence with at least 95% sequence identity to nucleotides 23-93 of SEQ ID NO: 202, nucleotides 23-157 of SEQ ID NO: 203, nucleotides 23-145 of SEQ ID NO: 613, or nucleotides 23-157 of SEQ ID NO: 614.