RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasY proteins, nucleic acids encoding the CasY proteins, and modified host cells comprising the CasY proteins and/or nucleic acids encoding same. CasY proteins are useful in a variety of applications, which are provided. The present disclosure provides CasY guide RNAs that bind to and provide sequence specificity to the CasY proteins, nucleic acids encoding the CasY guide RNAs, and modified host cells comprising the CasY guide RNAs and/or nucleic acids encoding same. CasY guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides methods of identifying a CRISPR RNA-guided endonuclease.
1. A composition comprising:
a) a CasY polypeptide, or a nucleic acid molecule encoding the CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7; and
b) a CasY guide RNA comprising:
i) a first segment that associates with the CasY polypeptide; and
ii) a second segment that binds to a target nucleic acid,
wherein the first and second segments are heterologous to one another, or,
one or more DNA molecules encoding the CasY guide RNA.
2. The composition of claim 1 , wherein the CasY polypeptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7.
3. The composition of claim 1 , wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 3.
4. The composition of claim 1 , wherein:
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 3 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 13;
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 14; or
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 6 or 7 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 15.
5. The composition of claim 1 , comprising one or more of: a buffer, a nuclease inhibitor, a lipid, and a protease inhibitor.
6. The composition of claim 1 , further comprising a second CasY guide RNA, or one or more DNA molecules encoding the second CasY guide RNA.
7. The composition of claim 6 , wherein the second segment of the first CasY guide RNA hybridizes to a first target sequence and wherein the second CasY guide RNA comprises a second segment that hybridizes to a second target sequence.
8. The composition of claim 7 , wherein the first target sequence and the second target sequence are in different target nucleic acids.
9. The composition of claim 7 , wherein the first target sequence and the second target sequence are in the same target nucleic acid.
10. The composition of claim 1 , wherein the CasY guide RNA is a hybrid DNA/RNA comprising DNA bases in addition to RNA bases.
11. A CRISPR-CAS system for detecting the presence of a target nucleic acid in a sample, the system comprising:
a) a CasY polypeptide, or a nucleic acid molecule encoding the CasY polypeptide, wherein the Cas Y polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7; and
b) a CasY guide RNA comprising:
i) a first segment that associates with the CasY polypeptide; and
ii) a second segment that binds to a target nucleic acid,
wherein the first and second segments are heterologous to one another,
or one or more DNA molecules encoding the CasY guide RNA,
wherein if the target nucleic acid is present in the sample and the sample is contacted with the system, the system can bind to the target nucleic acid.
12. One or more nucleic acid molecules encoding:
(a) a CasY guide RNA comprising:
i) a first segment that associates with a CasY polypeptide; and
ii) a second segment that binds to a target nucleic acid,
wherein the first and second segments are heterologous to one another; and
(b) the CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7.
13. The one or more nucleic acid molecules of claim 12 , wherein the CasY polypeptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7.
14. The one or more nucleic acid molecules of claim 12 , wherein:
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 3 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 13;
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 14; or
the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 6 or 7 and the CasY guide RNA comprises a nucleotide sequence having 80% or more identity with the sequence set forth in SEQ ID NO: 15.
15. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3 or 4; and
b) a CasY guide RNA comprising:
i) a first segment that associates with the CasY polypeptide; and
ii) a second segment that binds to a target nucleic acid,
wherein the first and second segments are heterologous to one another,
wherein the target nucleic acid is double-stranded DNA, and
wherein said contacting results in modification of the target nucleic acid by the CasY polypeptide.
16. The method of claim 15 , wherein said modification is cleavage of the target nucleic acid.
17. The method of claim 15 , wherein the target nucleic acid is genomic DNA or extrachromosomal DNA.
18. The method of claim 15 , wherein the target nucleic acid is in a cell-free composition.
19. The method of claim 15 , wherein contacting further comprises contacting a sample comprising the target nucleic acid and a non-target nucleic acid with the CasY polypeptide and the CasY guide RNA.
20. The method of claim 15 , wherein the target nucleic acid comprises a double stranded DNA comprising a complementary strand comprising a target sequence that hybridizes to the second segment and a non-complementary strand, wherein the target nucleic acid comprises a Protospacer Adjacent Motif (PAM) 5′ of the target sequence of the non-complementary strand.
21. The method of claim 20 , wherein the PAM sequence is 5′-TR-3′, wherein R is an A or G.
22. The method of claim 15 , wherein the contacting takes place in vitro and outside of a cell.
23. A CasY fusion polypeptide comprising: a CasY polypeptide fused to a heterologous polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3-7.
24. The CasY fusion polypeptide of claim 23 , wherein the CasY polypeptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3-7.
25. The CasY fusion polypeptide of claim 23 , wherein the heterologous polypeptide comprises horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galatosidase (GAL) glucose-6-phosphate dehydrogenase, beta-N-acetylgucosaminadase, beta-glucoronidase, invertase, Xanthine Oxidase, firefly luciferase, or glucose oxidase (GO).
26. A method of recognizing the presence of a target nucleic acid in a sample, the method comprising contacting the sample with:
a) a CasY polypeptide, wherein the CasY polypeptide comprises an amino acid sequence having 50% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 3 or 4; and
b) a CasY guide RNA comprising:
i) a first segment that associates with the CasY polypeptide; and
ii) a second segment that binds to a target nucleic acid,
wherein the first and second segments are heterologous to one another,
wherein the target nucleic acid is double-stranded DNA, and
wherein upon presence of the target nucleic acid in the sample, the contacting results in modification of the target nucleic acid by the CasY polypeptide.