RNA-guided endonuclease expressing algal strain 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 algal strain of the Class Trebouxiophyceae comprising:
a Cas protein expression construct comprising:
a Cas nuclease expression cassette;
a repressible CRE expression cassette comprising a Cre coding region; and
a selectable marker cassette;
a reporter expression cassette; and
two lox sites flanking the repressible CRE expression cassette and the selectable marker cassette.
2. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 1 , wherein the Cas protein is Cas9.
3. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 2 , further comprising an intron inserted into the Cre coding region of the Cre expression cassette.
4. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 3 , wherein the Cas9 expression cassette further comprises a promoter for constitutive expression of Cas9 nuclease.
5. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 4 , wherein the lox sites have the same orientation.
6. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 2 , wherein the alga is a member of the genus Parachlorella.
7. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 2 , wherein the Cas9 expression cassette comprises an N-terminal FLAG tag.
8. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 2 , wherein the selectable market cassette encodes blasticidin resistance.
9. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 8 , wherein the reporter cassette encodes a fluorescent protein.
10. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 1 , wherein the Cas protein is selected from the group consisting of: Cpf1, Csc2, Cas1, Cas2, Cas3, Cas4, and Cas10.
11. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 10 , further comprising an intron inserted into the Cre coding region of the Cre expression cassette.
12. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 11 , wherein the Cas protein expression cassette further comprises a promoter for constitutive expression of the Cas nuclease.
13. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 12 , wherein the lox sites have the same orientation.
14. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 1 , wherein the alga is a member of the genus Parachlorella.
15. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 10 , wherein the Cas protein expression cassette comprises an N-terminal FLAG tag.
16. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 10 , wherein the reporter cassette encodes a fluorescent protein.
17. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 10 , wherein the Cas protein is Cpf1 or Csc2.
18. The fully penetrant RNA-guided endonuclease expressing algal strain of claim 10 , wherein the Cas protein is selected from the group consisting of: Cas1, Cas3, and Cas4.