Compositions and methods for high efficiency in vivo genome editing

Patent metadata

Field	Value
Publication number	US-11339399-B2
Application number	US-201514986492-A
Country	US
Kind code	B2
Filing date	Dec 31, 2015
Priority date	Dec 31, 2014
Publication date	May 24, 2022
Grant date	May 24, 2022

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Synthetic Genomics Inc
• Viridos Inc

Inventors

• Verruto John
• Moellering Eric

Classifications

• C12N15/907

  in mammalian cells · CPC title

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N15/8209

  Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers · CPC title

• C12N9/1241 Primary

  Nucleotidyltransferases (2.7.7) · CPC title