RNA-guided nucleic acid modifying enzymes and methods of use thereof
The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.
1. One or more nucleic acid molecules encoding one or more of:
(a) a CasX guide RNA comprising an activator RNA and a targeter RNA, wherein the targeter RNA is heterologous to the activator RNA,
wherein one nucleic molecule comprises a first nucleotide sequence encoding the activator RNA and a second nucleotide sequence encoding the targeter RNA, wherein the first nucleotide sequence is heterologous to the second nucleotide sequence, or
wherein a first nucleic molecule comprises a nucleotide sequence encoding the activator RNA and a second nucleic molecule comprises a nucleotide sequence encoding the targeter RNA, wherein the first nucleic molecule is heterologous to the second nucleic molecule,
and
wherein the targeter RNA comprises (i) a guide sequence that hybridizes to a target nucleic acid, and (ii) a duplex-forming segment and the activator RNA hybridizes with the duplex-forming segment to form a double stranded RNA (dsRNA) duplex that binds a CasX polypeptide and guides the bound CasX polypeptide to the target nucleic acid;
(b) a CasX polypeptide, wherein the one nucleic acid molecule comprises a promoter operably linked to and heterologous to the sequence encoding the CasX polypeptide; and
(c) a CasX fusion polypeptide comprising a CasX polypeptide fused to a heterologous polypeptide, wherein the one nucleic acid molecule comprises a nucleotide sequence encoding the CasX fusion polypeptide.
2. The one or more nucleic acid molecules of claim 1 , wherein the CasX polypeptide comprises an amino acid sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
3. The one or more nucleic acid molecules of claim 1 , wherein the CasX guide RNA is a single guide RNA.
4. The one or more nucleic acid molecules of claim 1 , wherein the CasX guide RNA is a dual-guide RNA.
5. The one or more nucleic acid molecules of claim 1 , wherein the promoter is functional in a eukaryotic cell.
6. The one or more nucleic acid molecules of claim 1 , wherein the one or more nucleic acid molecules is one or more recombinant expression vectors.
7. The one or more nucleic acid molecules of claim 6 , wherein the one or more recombinant expression vectors are selected from: one or more adeno-associated viral vectors, one or more recombinant retroviral vectors, and one or more recombinant lentiviral vectors.
8. An isolated eukaryotic cell comprising one or more of:
a) a CasX polypeptide, or a nucleic acid molecule encoding the CasX polypeptide,
b) a CasX fusion polypeptide, or a nucleic acid molecule encoding the CasX fusion polypeptide, wherein the CasX fusion polypeptide comprises a CasX polypeptide fused to a heterologous polypeptide, and
c) a CasX guide RNA, or a nucleic acid molecule encoding the CasX guide RNA, wherein the CasX guide RNA comprises (i) a targeter sequence comprising a guide sequence that hybridizes to a target nucleic acid, and a duplex-forming segment; and (ii) an activator sequence that hybridizes with the duplex-forming segment of the targeter sequence to form a double stranded RNA(dsRNA) duplex that binds the CasX polypeptide and guides the bound CasX polypeptide to the target nucleic acid.
9. The isolated eukaryotic cell of claim 8 , comprising the nucleic acid molecule encoding the CasX polypeptide, wherein said nucleic acid molecule is integrated into the genomic DNA of the cell.
10. The isolated eukaryotic cell of claim 8 , wherein the eukaryotic cell is a plant cell, a mammalian cell, an insect cell, an arachnid cell, a fungal cell, a bird cell, a reptile cell, an amphibian cell, an invertebrate cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, or a human cell.
11. An isolated cell comprising a CasX fusion polypeptide comprising a CasX polypeptide fused to a heterologous polypeptide, or a nucleic acid molecule encoding the CasX fusion polypeptide, wherein the isolated cell is a prokaryotic cell.
12. The isolated cell of claim 11 , comprising the nucleic acid molecule encoding the CasX fusion polypeptide, wherein said nucleic acid molecule is integrated into the genomic DNA of the cell.
13. The one or more nucleic acid molecules of claim 1 , wherein the heterologous polypeptide is fused to the N- or the C-terminus of the CasX polypeptide.
14. The one or more nucleic acid molecules of claim 1 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies target DNA.
15. The one or more nucleic acid molecules of claim 1 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies a target polypeptide associated with a target nucleic acid.
16. The one or more nucleic acid molecules of claim 1 , wherein the heterologous polypeptide is an endosomal escape polypeptide.
17. The one or more nucleic acid molecules of claim 1 , wherein the heterologous polypeptide is a chloroplast transit peptide.
18. The one or more nucleic acid molecules of claim 1 , wherein the CasX guide RNA comprises a nucleotide sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity to the nucleotide sequence set forth in any one of SEQ ID NOs:11-17, 21-28, 31-34, 41-43, and 61.
19. The eukaryotic cell of claim 8 , wherein the CasX polypeptide comprises an amino acid sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.
20. The eukaryotic cell of claim 8 , wherein the CasX polypeptide can catalyze a double strand break in a target double-stranded DNA.
21. The eukaryotic cell of claim 8 , wherein the CasX polypeptide is a nickase that can cleave only one strand of a double-stranded target nucleic acid molecule.
22. The eukaryotic cell of claim 8 , wherein the CasX polypeptide is a catalytically inactive CasX polypeptide (dCasX).
23. The eukaryotic cell of claim 8 , wherein the CasX polypeptide comprises one or more mutations at a position corresponding to those selected from: D672, E769, and D935 of SEQ ID NO: 1.
24. The eukaryotic cell of claim 8 , wherein the heterologous polypeptide is fused to the N- or the C-terminus of the CasX polypeptide.
25. The eukaryotic cell of claim 8 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies target DNA.
26. The eukaryotic cell of claim 8 , wherein the heterologous polypeptide exhibits an enzymatic activity that modifies a target polypeptide associated with a target nucleic acid.
27. The eukaryotic cell of claim 8 , wherein the heterologous polypeptide is an endosomal escape polypeptide.
28. The eukaryotic cell of claim 8 , wherein the heterologous polypeptide is a chloroplast transit peptide.
29. The eukaryotic cell of claim 8 , wherein the guide RNA comprises a nucleotide sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 100% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 11-17, 21-28, 31-34, 41-43, and 61.
30. The one or more nucleic acid molecules of claim 1 , wherein the one or more nucleic acid molecules encode a detectable label.
31. The one or more nucleic acid molecules of claim 30 , wherein the detectable label comprises a fluorophore.