CRISPR-Cas12J 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 method of editing a target nucleic acid in a eukaryotic cell, wherein the target nucleic acid comprises double stranded DNA, the method comprising contacting the eukaryotic cell with:
a) a polypeptide comprising an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:126, or a nucleic acid encoding the polypeptide; and
b) a recombinant guide RNA, or a nucleic acid encoding the recombinant guide RNA, wherein the recombinant guide RNA comprises a guide sequence that hybridizes to a target sequence of a complementary strand of the target nucleic acid,
wherein a protospacer adjacent motif (PAM) is immediately 5′ of the target sequence of the non-complementary strand of the target nucleic acid, wherein the PAM is 5′-NTTN-3′, wherein T is thymine and N is any nucleotide.
2. The method of claim 1 , comprising contacting the eukaryotic cell ex vivo.
3. The method of claim 1 , comprising contacting the eukaryotic cell in vivo.
4. The method of claim 1 , wherein a nuclear localization signal (NLS) is fused to the N terminus of the polypeptide, the C terminus of the polypeptide, or both termini of the polypeptide.
5. The method of claim 1 , wherein the polypeptide is fused to a base editor, and wherein the method comprises modifying at least one nucleobase of the target nucleic acid.
6. The method of claim 1 , comprising inserting a DNA donor template into the target nucleic acid.
7. The method of claim 1 , comprising contacting the cell with an additional recombinant guide RNA.
8. The method of claim 1 , wherein the nucleic acid encoding the polypeptide is present in an expression vector.
9. The method of claim 8 , wherein the expression vector comprises the nucleic acid encoding the recombinant guide RNA.
10. The method of claim 8 , wherein the expression vector is an adenoviral associated viral vector.
11. The method of claim 1 , comprising contacting the cell with a lipid or lipid nanoparticle.
12. The method of claim 1 , wherein the nucleic acid encoding the polypeptide is a messenger RNA.
13. The method of claim 1 , wherein the polypeptide is 100% identical to SEQ ID NO: 126.
14. The method of claim 1 , wherein the eukaryotic cell is a human cell.
15. The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.
16. The method of claim 1 , wherein the eukaryotic cell is a post-mitotic cell.
17. The method of claim 1 , wherein the eukaryotic cell is selected from a myofibroblast, cardiomyocyte, myoblast, myocardial cell, cardiac myoblast, and a skeletal myoblast.
18. The method of claim 1 , wherein the eukaryotic cell is a hematopoietic stem cell.
19. The method of claim 1 , wherein the eukaryotic cell is selected from a monocyte, a macrophage, a T cell, a B cell, a natural killer cell, and a dendritic cell.
20. The method of claim 1 , wherein the PAM is 5′-VTTN-3′, wherein N is any nucleotide and V is selected from adenine, cytosine, and guanine.
21. The method of claim 1 , wherein the recombinant guide RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:179.
22. The method of claim 1 , wherein the recombinant guide RNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 179.
23. The method of claim 1 , wherein the eukaryotic cell is a liver cell.
24. The method of claim 1 , wherein the polypeptide is fused to a reverse transcriptase.
25. The method of claim 1 , wherein the polypeptide is fused to a heterologous polypeptide.