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 pharmaceutical composition comprising:
a) at least one expression vector encoding:
i) a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 120; and
ii) a recombinant guide RNA comprising:
1) A constant region comprising a first nucleotide sequence that is at least 80% identical to SEQ ID NO: 181; and
2) a targeting segment comprising a second nucleotide sequence
complementary to a target sequence on a target strand (TS) of a double-stranded DNA molecule, wherein the double-stranded DNA molecule comprises a 5′-NTTN-3′ protospacer adjacent motif (PAM) sequence positioned immediately 5′ of the target sequence on a non-target strand (NTS) of the double stranded DNA molecule, wherein T is thymine and N is any nucleotide, and
wherein a 3′ end of the first nucleotide sequence of the recombinant guide RNA is linked to a 5′ end of the second nucleotide sequence; and
b) a pharmaceutically acceptable excipient.
2. The pharmaceutical composition of claim 1 , wherein the polypeptide comprises an amino acid sequence of:
a) EEACKKFVREN (SEQ ID NO:251)
b) GPAIANIIAKSREFTEW (SEQ ID NO:252)
c) LNLIIKNAVN (SEQ ID NO:253)
d) VKVDNKNKNNL (SEQ ID NO:254)
e) RKNEIAKLNGEQE (SEQ ID NO:255)
f) GYLLQKPSPNKSIYCY (SEQ ID NO:256)
g) PGYVPKWQY (SEQ ID NO:257)
h) CVFDMRGLLRT (SEQ ID NO:258)
i) TVDVGQNNP (SEQ ID NO:259)
j) GEKFNCLKCG (SEQ ID NO:260) or
k) VAITAQSMPKP (SEQ ID NO:261).
3. The pharmaceutical composition of claim 1 , wherein the polypeptide does not begin with the amino acid sequence “MIS”.
4. The pharmaceutical composition of claim 1 , wherein the constant region is at least 90% identical to SEQ ID NO: 181.
5. The pharmaceutical composition of claim 1 , wherein the constant region is at least 95% identical to SEQ ID NO: 181.
6. The pharmaceutical composition of claim 1 , wherein the constant region has the nucleic acid sequence set forth in SEQ ID NO: 181.
7. The pharmaceutical composition of claim 1 , comprising a heterologous polypeptide that is fused to the polypeptide.
8. The pharmaceutical composition of claim 1 , comprising a subcellular localization sequence that is fused to the polypeptide.
9. The pharmaceutical composition of claim 1 , wherein the polypeptide is fused to a nuclear localization signal (NLS).
10. The pharmaceutical composition of claim 9 , wherein the NLS is fused to the N terminus of the polypeptide, the C terminus of the polypeptide, or both termini of the polypeptide.
11. The composition of claim 10 , wherein the NLS is fused to the N terminus of the polypeptide via an amide bond, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 49.
12. The composition of claim 10 , wherein the NLS is fused to the carboxyl terminus (C terminus) of the polypeptide, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 50.
13. The composition of claim 10 , 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.
14. The pharmaceutical composition of claim 1 , comprising a DNA donor template.
15. The pharmaceutical composition of claim 1 , comprising an additional recombinant guide RNA, wherein the sequence of the additional recombinant guide RNA is different from the sequence of the recombinant guide RNA.
16. The pharmaceutical composition of claim 1 , wherein the expression vector is an adenoviral vector, adenoviral-associated viral (AAV) vector, a lentiviral vector, or a retroviral vector.
17. The pharmaceutical composition of claim 1 , comprising one or more of: a buffer, a nuclease inhibitor, a protease inhibitor, and an adjuvant.
18. A method of editing a cell, the method comprising contacting the cell with the pharmaceutical composition of claim 1 .
19. The method of claim 18 , wherein the cell is an immune cell.
20. The method of claim 18 , wherein the cell is a T cell.
21. The method of claim 18 , wherein the cell is a stem cell.
22. The method of claim 18 , wherein the cell is a human cell.
23. The method of claim 18 , wherein the cell is a cancer cell.
24. The method of claim 18 , wherein the cell is in vivo.
25. The method of claim 18 , wherein the cell is ex vivo.
26. The method of claim 18 , wherein the cell is selected from: a cardiomyocyte, a muscle cell, a myofibroblast, a myoblast, and a myocardial cell.
27. The method of claim 18 , wherein the cell is a pluripotent stem cell or a hematopoietic stem cell.
28. The pharmaceutical composition of claim 1 , comprising a molecule selected from a lipid and a lipid nanoparticle.
29. The pharmaceutical composition of claim 1 , wherein the polypeptide is encoded by a first expression vector and the recombinant guide RNA is encoded by a second expression vector.
30. The pharmaceutical composition of claim 1 , wherein the polypeptide and the recombinant guide RNA are encoded by the expression vector.
31. The pharmaceutical composition of claim 30 , wherein the expression vector is an adenoviral-associated viral (AAV) vector.
32. The pharmaceutical composition of claim 1 , wherein the target sequence is a eukaryotic sequence.
33. The pharmaceutical composition of claim 1 , wherein the expression vector comprises a promoter that is operable in a mammalian cell.
34. The pharmaceutical composition of claim 1 , wherein the recombinant guide RNA is a CRISPR RNA (crRNA).
35. The pharmaceutical composition of claim 1 , wherein the length of the second nucleotide sequence is 14-20 nucleotides.
36. The pharmaceutical composition of claim 1 , wherein the polypeptide is a nuclease.
37. The pharmaceutical composition of claim 1 , wherein the 3′ end of the first nucleotide sequence of the recombinant guide RNA is directly linked to the 5′ end of the second nucleotide sequence.
38. The pharmaceutical composition of claim 1 , wherein the recombinant guide RNA comprises at least one nucleotide between the 3′ end of the first nucleotide sequence and the 5′ end of the second nucleotide sequence.