NOVEL CRISPR-ASSOCIATED (CAS) PROTEIN
A new CRISPR-associated (Cas) protein, termed “CasM,” is described, as well as polynucleotides encoding the same and methods of using CasM for site-specific genome engineering. CasM proteins are capable of targeting and cleaving single-stranded RNA.
1 . An isolated CasM protein capable of producing a single-strand break at an RNA target site when guided to the RNA target site by a cognate nucleic acid guide.
2 . (canceled)
3 . The CasM protein of claim 1 , wherein the protein comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOS: 37-44 or 45; an ortholog of the amino acid sequence of SEQ ID NOS: 37-44 and 45; and a variant of the amino acid sequence of SEQ ID NOS: 37-44 or 45.
4 . The CasM protein of claim 3 , wherein the protein comprises the amino acid sequence of SEQ ID NOS: 37-44 or 45.
5 . A complex comprising:
a CasM protein according to claim 1 ; and
a cognate nucleic acid guide.
6 . The complex of claim 5 , wherein the cognate nucleic acid guide comprises RNA.
7 . (canceled)
8 . The complex of claim 5 , wherein the cognate nucleic acid guide comprises a repeat sequence and a spacer sequence, wherein the repeat sequence and the spacer sequence do not naturally occur together.
9 . (canceled)
10 . An isolated polynucleotide encoding the CasM protein of claim 1 .
11 . The polynucleotide of claim 10 , wherein the polynucleotide comprises the sequence of SEQ ID NOS: 28-35 or 36.
12 . A modified polynucleotide encoding a CasM protein capable of producing a single strand break at a RNA target site when guided to the RNA target site by a cognate nucleic acid guide, wherein the polynucleotide is modified relative to its native sequence.
13 .- 15 . (canceled)
16 . The modified polynucleotide of claim 12 , wherein the polynucleotide comprises the sequence of SEQ ID NOS: 1-8 or 9.
17 . The modified polynucleotide of claim 12 , wherein the polynucleotide is modified for expression in a eukaryotic cell.
18 . (canceled)
19 . (canceled)
20 . The modified polynucleotide of claim 12 , wherein the polynucleotide comprises the sequence of SEQ ID NOS: 10-17 or 18.
21 . The modified polynucleotide of claim 12 , wherein the polynucleotide comprises a sequence encoding a N- or C-terminal nuclear localization signal sequence (NLS).
22 . The modified polynucleotide of claim 17 , wherein the polynucleotide is modified for expression in a plant cell.
23 . The modified polynucleotide of claim 22 , wherein the plant cell is a Zea mays cell.
24 . The modified polynucleotide of claim 23 , wherein the polynucleotide comprises the sequence of SEQ ID NOS: 20-26 or 27.
25 . A recombinant vector comprising:
a polynucleotide comprising a coding sequence for a CasM protein according to claim 1 ; and
at least one control element operably linked to said polynucleotide, whereby the CasM coding sequence in said polynucleotide is capable of being transcribed and translated in a host cell.
26 . (canceled)
27 . (canceled)
28 . A host cell comprising the recombinant vector of claim 25 .
29 .- 32 . (canceled)
33 . A method of directing a CasM protein to a selected nucleic acid target sequence, the method comprising:
contacting the selected nucleic acid target sequence with a complex according to claim 5 , whereby the CasM protein is delivered to the nucleic acid target sequence.
34 .- 40 . (canceled)