Methods and products for genetic engineering
The present invention relates to a virus-derived particle comprising one or more Cas protein(s), as well as to kits and methods using the same for altering a target nucleic acid.
1. A method of producing a retrovirus-derived particle comprising a Cas protein, the method comprising:
a) providing a cell that comprises a nucleic acid sequence encoding a fusion protein comprising (i) a GAG protein and (ii) a Cas protein;
b) culturing the cell under conditions that allow for production of a retrovirus-derived particle; and
c) separating the retrovirus-derived particle from the cell, wherein the retrovirus-derived particle comprises the Cas protein contained within said particle.
2. The method of claim 1 , wherein the cell comprises a nucleic acid sequence encoding the GAG protein and a nucleic acid sequence encoding a viral envelope protein, wherein the viral envelope protein is from the same virus as the GAG protein.
3. The method of claim 1 , wherein the cell comprises a nucleic acid sequence encoding the GAG protein and a nucleic acid sequence encoding a viral envelope protein, wherein the viral envelope protein is a pseudotyped protein that is from a different virus than the GAG protein.
4. The method of claim 3 , wherein the pseudotyped protein is a glycoprotein derived from another enveloped virus.
5. The method of claim 3 , wherein the Cas protein is Cas9 or a homolog or derivative thereof.
6. The method of claim 3 , wherein the Cas protein is Cas9.
7. The method of claim 3 , wherein the fusion protein is a cleavable fusion protein comprising a proteolysis cleavage site located between the GAG protein and the Cas protein.
8. The method of claim 7 , wherein the retrovirus-derived particle comprises the Cas protein as a free Cas protein resulting from the cleavage of the cleavable fusion protein.
9. A method of producing a retrovirus-derived particle comprising a Cas protein, the method comprising:
a) providing a cell that comprises a nucleic acid sequence encoding a GAG-Cas9 fusion protein;
b) culturing the cell under conditions that allow for production of a retrovirus-derived particle; and
c) separating the retrovirus-derived particle from the cell, wherein the retrovirus-derived particle comprises the Cas9 protein contained within said particle.
10. The method of claim 9 , wherein the GAG-Cas9 fusion protein is a cleavable fusion protein comprising a proteolysis cleavage site located between GAG and Cas9.
11. The method of claim 10 , wherein the retrovirus-derived particle comprises the Cas9 protein as a free Cas9 protein within the particle resulting from the cleavage of the cleavable fusion protein.
12. The method of claim 9 , wherein the cell comprises a nucleic acid sequence encoding one or more viral structural protein that is a Gag-Pro-Pol fusion protein.
13. The method of claim 12 , wherein the cell comprises a nucleic acid sequence encoding one or more viral envelope protein that is a pseudotyped protein from the same virus as the one or more viral structural protein.
14. The method of claim 9 , wherein the cell comprises a nucleic acid sequence encoding one or more viral structural protein and one or more viral envelope protein that is a pseudotyped protein that is from a different virus than the one or more viral structural protein.
15. The method of claim 14 , wherein the pseudotyped protein comprises a viral envelope protein selected from the group consisting of a VSV-G protein, a Measles virus F protein, an Influenza virus HA protein, a Moloney virus MLV-A protein, a Moloney virus MLV-E protein, a Baboon Endogenous retrovirus (BAEV) envelope protein, an Ebola virus glycoprotein and foamy virus envelope protein, or a combination thereof.
16. The method of claim 1 , wherein the retrovirus-derived particle is devoid of any encoding nucleic acids.
17. The method of claim 9 , wherein the retrovirus derived particle is devoid of any encoding nucleic acids.
18. The method of claim 1 , wherein the retrovirus-derived particle is a lentivirus-derived particle.
19. The method of claim 1 , wherein the retrovirus-derived particle is selected from the group consisting of Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus-derived vector particles, and Baboon endogenous virus-derived vector particles.
20. The method of claim 9 , wherein the retrovirus-derived particle is a lentivirus-derived particle.
21. The method of claim 9 , wherein the retrovirus-derived particle is selected from the group consisting of Moloney murine leukemia virus-derived vector particles, Bovine immunodeficiency virus-derived particles, Simian immunodeficiency virus-derived vector particles, Feline immunodeficiency virus-derived vector particles, Human immunodeficiency virus-derived vector particles, Equine infection anemia virus-derived vector particles, Caprine arthritis encephalitis virus-derived vector particles, and Baboon endogenous virus-derived vector particles.
22. The method of claim 1 , wherein the nucleic acid sequence encoding the fusion protein is placed under the control of a regulatory sequence for over-expression of the Cas protein in the cell.
23. The method of claim 9 , wherein the nucleic acid sequence encoding the GAG-Cas9 fusion protein is placed under the control of a regulatory sequence for over-expression of the Cas9 protein in the cell.
24. The method of claim 1 , wherein the cell further comprises one or more CRISPR-Cas guide RNAs, wherein the retrovirus-derived particle comprises the Cas protein complexed with a CRISPR-Cas system guide RNA.
25. The method of claim 24 , wherein the CRISPR-Cas system guide RNA comprises a) a first CRISPR-Cas system guide RNA that hybridizes with a first target sequence of a target nucleic acid, and b) a second CRISPR-Cas system guide RNA that hybridizes with a second target sequence of said target nucleic acid.
26. The method of claim 25 , wherein the retrovirus-derived particles comprise a CRISPR-Cas ribonucleoprotein complex comprising a complex of the Cas protein with a guide RNA.
27. The method of claim 9 , wherein the cell further comprises one or more CRISPR-Cas guide RNAs, wherein the retrovirus-derived particle comprises the Cas9 protein complexed with a CRISPR-Cas system guide RNA.
28. The method of claim 27 , wherein the CRISPR-Cas system guide RNA comprises a) a first CRISPR-Cas system guide RNA that hybridizes with a first target sequence of a target nucleic acid, and b) a second CRISPR-Cas system guide RNA that hybridizes with a second target sequence of said target nucleic acid.
29. The method of claim 27 , wherein the retrovirus-derived particle comprises a CRISPR-Cas9 ribonucleoprotein complex which is a complex of the Cas9 protein with a guide RNA.
30. The method of claim 24 , wherein the cell further comprises one or more targeting nucleic acids.
31. The method of claim 27 , wherein the cell further comprises one or more targeting nucleic acids.
32. The method of claim 1 , wherein the cell is a mammalian cell.
33. The method of claim 9 , wherein the cell is a mammalian cell.
34. A virus-derived particle produced by the method of claim 1 .
35. A virus-derived particle produced by the method of claim 8 .
36. A virus-derived particle produced by the method of claim 9 .
37. A virus-derived particle produced by the method of claim 11 .
38. The method of claim 1 , wherein the cell comprises a nucleic acid sequence encoding one or more viral structural protein that is a Gag-Pro-Pol fusion protein.
39. The method of claim 1 , wherein the Cas protein is Cpf1.
40. The method of claim 3 , wherein the pseudotyped protein comprises a viral envelope protein selected from the group consisting of a VSV-G protein, a Measles virus F protein, an Influenza virus HA protein, a Moloney virus MLV-A protein, a Moloney virus MLV-E protein, a Baboon Endogenous retrovirus (BAEV) envelope protein, an Ebola virus glycoprotein and foamy virus envelope protein, or a combination thereof.
41. The method of claim 1 , wherein the retrovirus-derived particle is a Moloney murine leukemia virus-derived vector particle.
42. The method of claim 1 , wherein the retrovirus-derived particle is a Human immunodeficiency virus-derived vector particle.
43. The method of claim 9 , wherein the retrovirus-derived particle is a Moloney murine leukemia virus-derived vector particle.
44. The method of claim 9 , wherein the retrovirus-derived particle is a Human immunodeficiency virus-derived vector particle.
45. The method of claim 1 , wherein the GAG protein is derived from Moloney murine leukemia virus.
46. The method of claim 1 , wherein the GAG protein is derived from Human immunodeficiency virus.
47. The method of claim 9 , wherein GAG is derived from Moloney murine leukemia virus.
48. The method of claim 9 , wherein GAG is derived from Human immunodeficiency virus.