ENZYMES WITH HEPN DOMAINS
The present disclosure provides for endonuclease enzymes having HEPN domains, as well as methods of using such enzymes or variants thereof.
1 . An engineered nuclease system comprising:
(a) an endonuclease comprising an HEPN domain, wherein said endonuclease is derived from an uncultivated microorganism; and
(b) an engineered guide ribonucleic acid structure configured to form a complex with said endonuclease comprising:
(i) a ribonucleic acid sequence configured to hybridize to a target ribonucleic acid sequence; and
(ii) a ribonucleic acid sequence configured to bind to said endonuclease.
2 . The engineered nuclease system of claim 1 , wherein said endonuclease comprises a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-15 and 62-84, or a variant thereof.
3 . The engineered nuclease system of claim 1 , wherein said endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease.
4 . The engineered nuclease system of claim 1 , wherein said endonuclease has less than 80% sequence identity to a Cas13b endonuclease.
5 . The engineered nuclease system of claim 1 , wherein said endonuclease comprises a sequence having at least about 75% sequence identity to any one of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 10, 11, 12, 13, or 15, or a variant thereof.
6 . The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises a repeat having a least 30 continuous nucleotides having at least about 80% sequence identity to any one of SEQ ID NOs: 21, 26, 30, 35, 41, 46, 50, 54, 60, 122, 123, 124, or 125.
7 . The engineered nuclease system of claim 1 , wherein said ribonucleic acid sequence configured to hybridize to said target ribonucleic acid sequence comprises at least about 18 to about 26 nucleotides.
8 . The engineered nuclease system of claim 6 , wherein said engineered guide ribonucleic acid structure is provided as a sequence comprising:
(i) a first copy of said repeat;
(ii) said ribonucleic acid sequence configured to hybridize to said target ribonucleic acid sequence; and
(iii) a second copy of said repeat.
9 . The engineered nuclease system of claim 1 , wherein said engineered guide ribonucleic acid structure comprises a sequence having at least about 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 36, 37, 55, or 61.
10 .- 24 . (canceled)
25 . An engineered guide ribonucleic acid polynucleotide comprising:
(a) a ribonucleic acid (RNA)-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target RNA molecule; and
(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 one another with intervening nucleotides, and
wherein said engineered guide ribonucleic acid polynucleotide is configured to form a complex with an endonuclease comprising a sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-15 and 62-84, or a variant thereof and target said complex to said target sequence of said target RNA molecule.
26 . The engineered guide ribonucleic acid polynucleotide of claim 25 , wherein said RNA-targeting segment is positioned 5′ of both of said two complementary stretches of nucleotides.
27 . A deoxyribonucleic acid polynucleotide encoding the engineered guide ribonucleic acid polynucleotide of claim 25 .
28 .- 36 . (canceled)
37 . A method for binding, cleaving, marking, or modifying a single-stranded ribonucleic acid polynucleotide, comprising:
contacting said single-stranded ribonucleic acid polynucleotide with a class 2, type VI endonuclease in complex with an engineered guide ribonucleic acid structure configured to bind to said endonuclease and target said class 2, type VI endonuclease to a target ribonucleic acid sequence and said single-stranded ribonucleic acid polynucleotide.
38 . The method of claim 37 , wherein said single-stranded ribonucleic acid polynucleotide comprises a protospacer flanking site (PFS).
39 . The method of claim 38 , wherein said PFS comprises GTT.
40 . The method of claim 37 , wherein said single-stranded ribonucleic acid polynucleotide comprises a sequence complementary to a sequence of said engineered guide ribonucleic acid structure and a PFS.
41 . The method of claim 38 , wherein said PFS is adjacent to said sequence complementary to said sequence of said engineered guide ribonucleic acid structure.
42 . The method of claim 37 , wherein said single single-stranded ribonucleic acid polynucleotide does not comprise a protospacer flanking site (PFS).
43 . The method of claim 37 , wherein said class 2, type VI endonuclease is not a Cas9 endonuclease, a Cas14 endonuclease, a Cas12a endonuclease, a Cas12b endonuclease, a Cas12c endonuclease, a Cas12d endonuclease, a Cas12e endonuclease, a Cas13a endonuclease, a Cas13b endonuclease, a Cas13c endonuclease, or a Cas13d endonuclease.
44 . The method of claim 37 , wherein said single-stranded ribonucleic acid polynucleotide is a eukaryotic, plant, fungal, mammalian, rodent, or human single-stranded ribonucleic acid polynucleotide.
45 .- 63 . (canceled)