Thermostable RNA-guided endonucleases and methods of use thereof
The present disclosure provides RNA-guided endonucleases, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided endonuclease of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided endonuclease of the present disclosure and a guide RNA.
1. An RNA-guided endonuclease that comprises:
a) an amino acid sequence having at least 95% amino acid sequence identity to amino acids 1-994 of the amino acid sequence set forth in SEQ ID NO: 1; and
b) a heterologous protospacer adjacent motif (PAM) interacting (PI) domain comprising an amino acid sequence having at least 95% amino acid sequence identity to the following amino acid sequence: IIKTAGGEEIKIKDLFAYYKTIHSGTAGLELVSHDCSFSLSGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETIRPLQSTRD (SEQ ID NO: 4), wherein the PI domain is heterologous to the amino acid sequence of part a).
2. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease is enzymatically active in a temperature range of from 15° C. to 75° C.
3. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease binds to a target nucleic acid comprising a PAM comprising a GMAA sequence, wherein M is A or C.
4. The RNA-guided endonuclease of claim 1 , wherein the heterologous PI domain comprises an amino acid sequence having at least 99% amino acid sequence identity to the following amino acid sequence:
(SEQ ID NO: 4)
IIKTAGGEEIKIKDLFAYYKTIHSGTAGLELVSHDCSFSLSGVGSRTLKRF
EKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETIRPLQSTRD.
5. The RNA-guided endonuclease of claim 1 , wherein the heterologous PI domain comprises an amino acid sequence having less than 83% amino acid sequence identity to the following amino acid sequence:
(SEQ ID NO: 5)
TVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRF
EKYQVDVLGNIYKVRGEKRVGLASSAHSKPGKTIRPLQSTRD.
6. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease has a length of from 1050 amino acids to 1120 amino acids.
7. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease has a length of from 1080 amino acids to 1095 amino acids.
8. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease comprises an amino acid sequence having at least 99% amino acid sequence identity to amino acids 1-994 of the amino acid sequence set forth in SEQ ID NO: 1.
9. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease comprises: a) a substitution of amino acid Asp-8; b) a substitution of amino acid His-582; or c) substitution of both Asp-8 and His-582, based on the amino acid numbering of the amino acid sequence of SEQ ID NO:1.
10. A fusion polypeptide comprising:
a) an RNA-guided endonuclease that comprises:
i) an amino acid sequence having at least 95% amino acid sequence identity to amino acids 1-994 of the amino acid sequence set forth in SEQ ID NO: 1; and
ii) a heterologous protospacer adjacent motif (PAM) interacting (PI) domain comprising an amino acid sequence having at least 95% amino acid sequence identity to the following amino acid sequence: IIKTAGGEEIKIKDLFAYYKTIHSGTAGLELVSHDCSFSLSGVGSRTLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKTGETIRPLQSTRD (SEQ ID NO: 4), wherein the PI domain is heterologous to the amino acid sequence of part i); and
b) a heterologous fusion partner polypeptide.
11. A cell comprising the RNA-guided endonuclease of claim 1 .
12. A ribonucleoprotein (RNP) complex comprising:
a1) the RNA-guided endonuclease of claim 1 ; and
b1) a guide RNA comprising:
i) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target nucleotide sequence in a target nucleic acid; and
ii) a protein-binding segment that binds to and activates the thermostable RNA-guided endonuclease, wherein the protein-binding segment comprises a duplex-forming linker segment and a tracrRNA.
13. A method of modifying a target DNA, the method comprising contacting the target DNA with the RNP complex of claim 12 .
14. The RNA-guided endonuclease of claim 1 , wherein the RNA-guided endonuclease comprises:
a) an amino acid sequence having amino acids 1-994 of the sequence set forth in SEQ ID NO: 1; and
b) a heterologous PI domain comprising the amino acid sequence of SEQ ID NO: 4.
15. The fusion polypeptide of claim 10 , wherein the RNA-guided endonuclease comprises an amino acid sequence having at least 99% amino acid sequence identity to amino acids 1-994 of the amino acid sequence set forth in SEQ ID NO: 1.
16. The fusion polypeptide of claim 10 , wherein the heterologous PI domain comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.