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 retrovirus-derived particle comprising one or more Cas protein(s) wherein the one or more Cas protein is contained within an inside of said particle as a fusion protein between (i) a viral structural protein and (ii) the one or more Cas protein(s), wherein the retrovirus-derived particle is devoid of any encoding nucleic acid, and wherein the one or more Cas protein(s) is complexed with one or more CRISPR-Cas system guide RNA(s).
2. The retrovirus-derived particle according to claim 1 , wherein said Cas protein is Cas9 or an homolog or a derivative thereof.
3. The retrovirus-derived particle according to claim 1 , wherein the CRISPR-Cas system guide RNAs comprise:
a first CRISPR-Cas system guide RNA that hybridizes with a first target sequence of a target nucleic acid, and
a second CRISPR-Cas system guide RNA that hybridizes with a second target sequence of said target nucleic acid.
4. The retrovirus-derived particle according to claim 1 , which is a lentivirus-derived particle.
5. The retrovirus-derived particle according to claim 1 , which 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 particle, and Baboon endogenous virus-derived vector particles.
6. A composition for altering a target nucleic acid in a eukaryotic cell, which composition comprises at least one retrovirus-derived particle as defined in claim 1 .
7. The composition according to claim 6 , further comprising one or more transduction helper compounds.
8. A kit for preparing retrovirus-derived particles for altering a target nucleic acid in a eukaryotic cell comprising:
a nucleic acid comprising an expression cassette encoding a GAG-Cas fusion protein,
a nucleic acid comprising one or more expression cassette(s) encoding virus-derived assembly protein(s), and
one or more nucleic acid(s) encoding a CRISPR-Cas system Guide RNA.
9. A cell line for producing a retrovirus-derived particle according to claim 1 , comprising:
one or more nucleic acids encoding proteins required for forming said virus-derived particle,
a nucleic acid comprising an expression cassette encoding a GAG-Cas fusion protein, and
nucleic acid(s) encoding one or more CRISPR guide RNA(s).
10. An in vitro or ex vivo method for altering, in at least one eukaryotic cell, a target nucleic acid comprising at least one target sequence, comprising the steps of:
a) contacting the at least one eukaryotic cell with one or more retrovirus-derived particles as defined in claim 1 , wherein said step of contacting is performed under conditions which permit the virus-derived particles to infect the at least one eukaryotic cell,
and
b) collecting eukaryotic cells having an altered target nucleic acid.
11. The retrovirus-derived particle according to claim 1 , wherein the Cas protein is present as a cleavable fusion protein comprising a proteolysis cleavage site located between the viral structural protein moiety and the Cas protein moiety.
12. The retrovirus-derived particle according to claim 1 , wherein the viral structural protein is a retroviral gag protein or a protein fragment thereof.
13. The retrovirus-derived particle of claim 1 , wherein the Cas protein is Cas9.
14. The retrovirus-derived particle of claim 13 , wherein the viral structural protein is a retroviral gag protein or a protein fragment thereof.
15. The retrovirus-derived particle according to claim 2 , wherein the Cas protein is present as a cleavable fusion protein comprising a proteolysis cleavage site located between the viral structural protein moiety and the Cas protein moiety.
16. The retrovirus-derived particle according to claim 13 , wherein the Cas protein is present as a cleavable fusion protein comprising a proteolysis cleavage site located between the viral structural protein moiety and the Cas protein moiety.
17. The retrovirus-derived particle according to claim 2 , which is a lentivirus-derived particle.
18. The retrovirus-derived particle according to claim 11 , which is a lentivirus-derived particle.
19. The retrovirus-derived particle according to claim 12 , which is a lentivirus-derived particle.
20. The retrovirus-derived particle according to claim 13 , which is a lentivirus-derived particle.
21. A composition comprising at least one retrovirus-like particle of claim 20 .
22. The cell line of claim 9 , wherein the virus-derived particle is a lentivirus-derived particle.
23. The cell line of claim 9 , wherein the Cas protein is Cas9.
24. The cell line of claim 22 , wherein the Cas protein is Cas9.