CRISPR-Cas effector polypeptides and methods of use thereof
The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.
1. A composition comprising:
a) a polypeptide consisting of the amino acid sequence of SEQ ID NO:120; and
b) a recombinant guide RNA comprising;
i. a nucleotide sequence that is complementary to a target sequence of a target nucleic acid and
ii. a region that associates with the polypeptide to form a guide RNA-polypeptide complex, wherein (i) is heterologous to (ii).
2. The composition of claim 1 , wherein the recombinant guide RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:181.
3. The composition of claim 1 , wherein the recombinant guide RNA comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:181.
4. The composition of claim 1 , comprising a nuclear localization signal (NLS) fused to the amino terminus (N terminus), the carboxyl terminus (C terminus), or both termini of the polypeptide.
5. The composition of claim 4 , wherein the NLS is fused to the N terminus of the polypeptide, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO:49 or SEQ ID NO:50.
6. The composition of claim 4 , wherein the NLS is fused to the C terminus of the polypeptide, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO:49 or SEQ ID NO:50.
7. The composition of claim 4 , wherein a first NLS comprising the amino acid sequence set forth in SEQ ID NO: 49 is fused to the N terminus of the polypeptide and wherein a second NLS comprising the amino acid sequence set forth in SEQ ID NO: 50 is fused to the C terminus of the polypeptide.
8. The composition of claim 1 , comprising a DNA donor template.
9. The composition of claim 1 , comprising a lipid, a liposome, a vector, or a particle.
10. The composition of claim 1 , comprising one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor.
11. The composition of claim 1 , comprising an additional recombinant guide RNA, wherein said additional recombinant guide RNA comprises a region that is complementary to a second target sequence.
12. The composition of claim 1 , comprising the target nucleic acid, wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) adjacent to said target sequence, wherein the PAM comprises the sequence 5′-NTTN-3′, wherein N is any nucleotide and T is thymine.
13. The composition of claim 1 , comprising a heterologous polypeptide that is fused to the polypeptide.
14. The composition of claim 1 , comprising a pharmaceutically acceptable excipient.
15. A population of cells comprising the composition of claim 1 .
16. The population of cells of claim 15 , wherein the population of cells comprises a cancer cell, an animal cell, a HEK293 cell, or an immune cell.
17. The population of cells of claim 16 , wherein the animal cell is a human cell.
18. A method of editing a gene comprising contacting a cell comprising the gene with the composition of claim 1 .
19. The method of claim 18 , wherein said editing comprises modifying the nucleotide sequence of the gene.
20. A method of producing a recombinant protein, comprising culturing the population of cells of claim 15 .
21. The composition of claim 1 , wherein the polypeptide begins with the amino acid sequence MIK.
22. The composition of claim 1 , wherein the recombinant guide RNA comprises one or more of a base modification, a sugar modification, and a backbone modification.
23. The composition of claim 1 , wherein the target sequence is a eukaryotic sequence.
24. The composition of claim 12 , wherein the target sequence is a eukaryotic sequence.
25. The composition of claim 12 , wherein the target nucleic acid comprises: (i) a target strand (TS) and (ii) a non-target strand (NTS) comprising the PAM wherein the PAM is 5′ to the target sequence.
26. The composition of claim 2 , wherein the nucleotide sequence that is at least 80% identical to SEQ ID NO: 181 is in a 5′ orientation with respect to the nucleotide sequence that is complementary to the target sequence.