Endoribonuclease and methods of use thereof
The present disclosure provides variant Cas endoribonucleases, nucleic acids encoding the variant Cas endoribonucleases, and host cells genetically modified with the nucleic acids. The variant Cas endoribonucleases find use in a variety of applications, which are also provided. The present disclosure also provides methods of detecting a specific sequence in a target polyribonucleotide; and methods of regulating production of a target RNA in a eukaryotic cell.
1. A variant Cas endoribonuclease comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:19, wherein the endoribonuclease comprises an amino acid substitution at a histidine residue such that the variant Cas endoribonuclease is enzymatically inactive in the absence of imidazole, and wherein the variant Cas endoribonuclease is activatable in the presence of imidazole.
2. The variant Cas endoribonuclease of claim 1 , wherein the amino acid substitution is a substitution at His37.
3. The variant Cas endoribonuclease of claim 1 , wherein the variant Cas endoribonuclease comprises a moiety that provides a detectable signal.
4. The variant Cas endoribonuclease of claim 1 , wherein the endoribonuclease is immobilized on an insoluble support.
5. The variant Cas endoribonuclease of claim 1 , wherein the variant Cas endoribonuclease binds an RNA substrate comprising a nucleotide sequence selected from:
(SEQ ID NO: 1)
5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;
(SEQ ID NO: 2)
5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;
(SEQ ID NO: 3)
5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;
(SEQ ID NO: 4)
5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;
(SEQ ID NO: 5)
5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;
(SEQ ID NO: 6)
5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;
(SEQ ID NO: 7)
5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′;
(SEQ ID NO: 8)
5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;
(SEQ ID NO: 9)
5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;
(SEQ ID NO: 10)
5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;
(SEQ ID NO: 11)
5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;
and
(SEQ ID NO: 12)
5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′.
6. A kit for purifying a target RNA present in a mixed population of nucleic acids, the kit comprising:
the variant Cas endoribonuclease of claim 1 .
7. The kit of claim 6 , further comprising a recombinant expression vector comprising, in order from 5′ to 3′ and in operable linkage:
a) a nucleotide sequence encoding an RNA substrate that is specifically bound by the variant Cas endoribonuclease of claim 1 ; and
b) a multiple cloning site suitable for insertion of a nucleic acid encoding the target RNA.
8. The kit of claim 7 , wherein the nucleotide sequence encoding the RNA substrate is operably linked to a promoter.
9. The kit of claim 7 , wherein the RNA substrate comprises a nucleotide sequence selected from:
(SEQ ID NO: 1)
5′-GUUGCAAGGGAUUGAGCCCCGUAAGGGGAUUGCGAC-3′;
(SEQ ID NO: 2)
5′-GUUGCAAACCUCGUUAGCCUCGUAGAGGAUUGAAAC-3′;
(SEQ ID NO: 3)
5′-GGAUCGAUACCCACCCCGAAGAAAAGGGGACGAGAAC-3′;
(SEQ ID NO: 4)
5′-GUCGUCAGACCCAAAACCCCGAGAGGGGACGGAAAC-3′;
(SEQ ID NO: 5)
5′-GAUAUAAACCUAAUUACCUCGAGAGGGGACGGAAAC-3′;
(SEQ ID NO: 6)
5′-CCCCAGUCACCUCGGGAGGGGACGGAAAC-3′;
(SEQ ID NO: 7)
5′-GUUCCAAUUAAUCUUAAACCCUAUUAGGGAUUGAAAC-3′;
(SEQ ID NO: 8)
5′-GUCGCCCCCCACGCGGGGGCGUGGAUUGAAAC-3′;
(SEQ ID NO: 9)
5′-CCAGCCGCCUUCGGGCGGCUGUGUGUUGAAAC-3′;
(SEQ ID NO: 10)
5′-GUCGCACUCUACAUGAGUGCGUGGAUUGAAAU-3′;
(SEQ ID NO: 11)
5′-UGUCGCACCUUAUAUAGGUGCGUGGAUUGAAAU-3′;
and
(SEQ ID NO: 12)
5′-GUCGCGCCCCGCAUGGGGCGCGUGGAUUGAAA-3′.
10. The kit of claim 7 , wherein the recombinant expression vector comprises, inserted into the multiple cloning site, a nucleotide sequence encoding the target RNA.
11. A method of isolating a target RNA present in a mixed population of nucleic acids, the method comprising:
a) contacting a mixed population of nucleic acids with the variant Cas endoribonuclease of claim 1 , where the variant Cas endoribonuclease is immobilized on an insoluble support, wherein the mixed population of nucleic acids comprises a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the immobilized variant Cas endoribonuclease, forming a tagged target RNA-immobilized variant Cas endoribonuclease complex, wherein said contacting is carried out in a binding solution lacking imidazole;
b) adding imidazole to the binding solution to a final concentration of from about 100 mM to about 500 mM, thereby forming a reactivation solution that enzymatically reactivates the immobilized variant Cas endoribonuclease, wherein the reactivated immobilized variant Cas endoribonuclease cleaves the target RNA from the tag; and
c) collecting the released target RNA.
12. The method of claim 11 , further comprising a wash step carried out after step (a) and before step (b).
13. A method of isolating a polypeptide that binds a target RNA, the method comprising:
a) contacting an immobilized complex with a liquid solution comprising a polypeptide that binds the target RNA, wherein the immobilized complex comprises the variant Cas endoribonuclease of claim 1 and a tagged target RNA comprising a recognition nucleotide sequence that is specifically bound by the variant Cas endoribonuclease, wherein said contacting results in binding of the polypeptide to the target RNA, wherein said contacting is carried out in a binding solution lacking imidazole; and
b) eluting the bound polypeptide.