Method for increasing the efficiency of double-strand-break induced mutagenesis

Patent metadata

Field	Value
Publication number	US-9540623-B2
Application number	US-201214131210-A
Country	US
Kind code	B2
Filing date	Jul 3, 2012
Priority date	Jul 8, 2011
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

The present invention relates to a method for increasing double-strand-break induced mutagenesis at a genomic locus of interest in a cell, thereby giving new tools for genome engineering, including therapeutic applications and cell line engineering. More specifically, the present invention concerns the combined use of TALEN or meganucleases with TREX2, especially under the form of single-chain proteins.

First claim

The invention claimed is:

1. A method for increasing double-strand-break induced mutagenesis at a genomic locus of interest in a cell comprising the steps of:
(i) identifying at said genomic locus of interest at least one DNA target sequence cleavable by one natural or engineered rare-cutting endonuclease;
(ii) expressing said rare-cutting endonuclease in the cell together with a polynucleotide encoding a fusion protein that comprises:
a first polypeptide that is a human TREX2 exonuclease protomer of SEQ ID NO: 26 or functional mutant thereof that shares at least 80% identity with the human TREX2 exonuclease protomer;
a second polypeptide that is a human TREX2 exonuclease protomer of SEQ ID NO: 26 or functional mutant thereof that shares at least 80% identity with the human TREX2 exonuclease protomer; and
a peptidic linker connecting said first and second polypeptides;
(iii) thereby obtaining, by expression of said fusion protein in said cell, increased double strand-break-induced mutagenesis at said genomic locus of interest.

2. The method of claim 1 , wherein the polynucleotide further encodes the rare-cutting endonuclease.

3. The method of claim 1 , wherein the rare-cutting endonuclease is encoded by a second polynucleotide.

4. The method of claim 2 , wherein the rare-cutting endonuclease is a meganuclease.

5. The method of claim 2 , wherein the rare-cutting endonuclease is selected from the group consisting of I-SceI, I-ChuI, I-CreI, I-CsmI, PI-SceI, PI-TliI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI, PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-MflI, PI-MgaI, PI-MgoI, PI-MinI, PI-MkaI, PI-MleI, PI-MmaI, PI-MshI, PI-MsmI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, and I-MsoI.

6. The method of claim 4 , wherein the meganuclease shares at least 80% identity with residues 1-152 of the natural I-CreI LAGLIDADG meganuclease of SEQ ID NO: 35.

7. The method of claim 4 , wherein the meganuclease is the natural I-CreI LAGLIDADG meganuclease of SEQ ID NO: 35.

8. The method of claim 2 , wherein the rare-cutting endonuclease is a TAL nuclease (TALEN).

9. The method of claim 2 , wherein said cell is a plant cell.

10. The method of claim 2 , wherein said cell is a mammalian cell.

11. The method of claim 2 , wherein said cell is a human cell.

12. The method of claim 3 , wherein the rare-cutting endonuclease is a meganuclease.

13. The method of claim 3 , wherein the rare-cutting endonuclease is selected from the group consisting of I-SceI, I-ChuI, I-CreI, I-CsmI, PI-SceI, PI-TliI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI, PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-MflI, PI-MgaI, PI-MgoI, PI-MinI, PI-MkaI, PI-MleI, PI-MmaI, PI-MshI, PI-MsmI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, and I-MsoI.

14. The method of claim 12 , wherein the meganuclease shares at least 80% identity with residues 1-152 of the natural I-CreI LAGLIDADG of SEQ ID NO: 35.

15. The method of claim 12 , wherein the meganuclease is the natural I-CreI LAGLIDADG meganuclease of SEQ ID NO: 35.

16. The method of claim 3 , wherein the rare-cutting endonuclease is a TAL nuclease (TALEN).

17. The method of claim 3 , wherein said cell is a plant cell.

18. The method of claim 3 , wherein said cell is a mammalian cell.

19. The method of claim 3 , wherein said cell is a human cell.

20. The method of claim 1 , wherein the first and second polypeptides are functional mutants of the human TREX2 exonuclease protomer of SEQ ID NO: 26 that share at least 90% identity with the human TREX2 exonuclease protomer.

21. The method of claim 1 , wherein the first and second polypeptides are functional mutants of the human TREX2 exonuclease protomer of SEQ ID NO: 26 that share at least 95% identity with the human TREX2 exonuclease protomer.

22. The method of claim 1 , wherein the first and second polypeptides are functional mutants of the human TREX2 exonuclease protomer of SEQ ID NO: 26 that share at least 97% identity with the human TREX2 exonuclease protomer.

Assignees

• Silva George H
• Macmaster Rachel
• Cellectis

Inventors

• Silva George H
• Macmaster Rachel

Classifications

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/82

  for plant cells {, e.g. plant artificial chromosomes (PACs)} · CPC title