Compositions and methods for high efficiency in vivo genome editing
The present invention provides cell lines for high efficiency genome editing using cas/CRISPR systems, methods of generating such cells lines, and methods of generating mutations in the genome of an organism using such cell lines.
1. A fully penetrant RNA-guided endonuclease-expressing Nannochloropsis or Parachlorella algal strain comprising, a nucleic acid sequence encoding a heterologous RNA-guided endonuclease;
a nucleic acid sequence encoding a nuclear localization signal linked to the nucleic acid sequence encoding the heterologous RNA-guided endonuclease; and
a nucleic acid sequence encoding a fluorescent detectable protein and;
wherein the fully penetrant algal strain exhibits culture-wide expression of the heterologous RNA-guided endonuclease, as indicated by a single fluorescent peak in a flow cytometry histogram.
2. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 1 , wherein the RNA-guided endonuclease is a Cas nuclease.
3. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 2 , wherein the RNA-guided endonuclease is a Cas9, Cpf1, C2c1, C2c2, or C2c3 nuclease.
4. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 1 , wherein the strain has a targeted mutation rate of at least 50% using a gRNA and donor fragment that comprises a selectable marker.
5. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 1 , wherein the fully penetrant RNA-guided endonuclease-expressing algal strain does not include a fluorescent protein gene.
6. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 1 , wherein the RNA-guided endonuclease-expressing algal strain further comprises an exogenous gene encoding a site-specific recombinase.
7. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 6 , wherein the site-specific recombinase is cre, frt, or dre.
8. A fully penetrant RNA-guided endonuclease-expressing algal strain according to claim 7 , wherein the exogenous gene encoding a site-specific recombinase is operably linked to an inducible promoter.
9. A method of altering the genome of a Nannochloropsis or Parachlorella algal cell, wherein the method comprises:
introducing at least one guide RNA or at least one construct for expressing at least one guide RNA into a Nannochloropsis or Parachlorella algal strain according to claim 1 , wherein the guide RNA targets a site in the genome of the cell; and
screening cells transformed with the guide RNA for alteration of the targeted site in the genome.
10. A method according to claim 9 , wherein the at least one guide RNA is a chimeric guide RNA.
11. A method according to claim 9 , wherein the at least one guide RNA is a crRNA.
12. A method according to claim 9 , wherein the fully penetrant RNA-guided endonuclease-expressing algal strain further comprises a construct encoding a tracrRNA.
13. A method according to claim 9 , wherein the method further comprises introducing a tracrRNA into the fully penetrant RNA-guided endonuclease-expressing algal strain.
14. A method according to claim 9 , wherein the method further comprises transforming a donor DNA into the fully penetrant RNA-guided endonuclease-expressing algal strain.
15. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 1 wherein the heterologous RNA-guided endonuclease is Cas9 and the fluorescent detectable protein is selected from the group consisting of: a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, and a cyan fluorescent protein.
16. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 15 wherein the fluorescent detectable protein is green fluorescent protein.
17. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 1 wherein the strain has a genome editing efficiency of at least 50%.
18. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 17 wherein the strain has a genome editing efficiency of at least 80%.
19. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 1 wherein the algal strain is of the genus Nannochloropsis.
20. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 1 is of the genus Parachlorella.
21. The method of claim 9 wherein the algal cell is of the genus Nannochloropsis.
22. The method of claim 9 wherein the algal cell is of the genus Parachlorella.
23. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim further comprising a nucleic acid sequence encoding a selectable marker.
24. The fully penetrant RNA-guided endonuclease-expressing algal strain of claim 1 wherein the fluorescent detectable protein is green fluorescent protein.