ENGINEERED LANDING PADS FOR GENE TARGETING IN PLANTS

• US 20110191899A1
• Filed: 01/21/2011
• Published: 08/04/2011
• Est. Priority Date: 01/22/2010
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Inventors: Miles, Rebbeca R., Blue, Ryan C., Murray, Michael G., Corbin, David, Webb, Steven R., Ainley, William M.
• Granted Patent: US 8,802,921 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology

Abstract

A method for producing a transgenic plant includes providing a nucleic acid molecule comprising at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell, and at least two zinc finger nuclease recognition sites, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell flank the at least two zinc finger nuclease recognition sites. A plant cell or tissue having the nucleic acid molecule stably integrated into the genome of the plant cell is transformed. A plant is regenerated from the plant cell. Transgenic plants are produced by the method. Seeds are produced by the transgenic plants.

Claims

1. A method for producing a transgenic plant or plant tissue, the method comprising:

providing a nucleic acid molecule comprising at least two regions of nucleic acid sequence that substantially lack sequence homology with genomic DNA of a plant cell, and at least one targeting endonuclease recognition site, wherein the at least two regions of nucleic acid sequence that lack substantial sequence homology with genomic DNA of the plant cell flank the at least one targeting endonuclease recognition site;

transforming the plant cell or plant tissue comprising the plant cell with the nucleic acid molecule, wherein the nucleic acid molecule is stably integrated into the genome of the plant cell; and

generating plant tissue or regenerating a plant from the plant cell transformed with the nucleic acid molecule.

2. The method of claim 1, wherein the nucleic acid molecule further comprises non-identical restriction sites.

3. The method of claim 2, wherein the non-identical restriction sites comprise compatible single-stranded ends that allow concatemerization of multiple ELPs.

4. The method of claim 1, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell do not comprise an open reading frame.

5. The method of claim 1, wherein the at least one region of nucleic acid sequence that lacks sequence homology with genomic DNA of the plant cell does not comprise an open reading frame.

6. The method of claim 1, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell are from about 50 bp to about 3 kb.

7. The method of claim 1, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell are selected from one of more of the group consisting of Sequence ID NOs 1 through 10.

8. The method of claim 1, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell together with targeting endonucleases comprise Sequence IDs 15 and 16.

9. The method of claim 1, wherein the at least two regions of nucleic acid sequence that lack sequence homology with genomic DNA of the plant cell together with targeting endonucleases comprise Sequence IDs 15 and 16 incorporated into plant transformation vectors, pDAB104137, pDAB104139, pDAB104141, or pDAB104143, used for transforming monocots, dicots, including canola.

10. The method according to claim 1, wherein the targeting endonuclease is a zinc finger nuclease.

11. The method of claim 1, wherein the nucleic acid molecule is stably integrated randomly in the genome of the plant cell.

12. The method of claim 1, wherein the nucleic acid molecule is stably integrated at one or more known target sites in the genome of the plant cell.

13. The method of claim 1, wherein the nucleic acid molecule is stably integrated into an amplified region of a chromosome or a minichromosome.

14. The transgenic plant tissue or plant produced by the method of claim 1.

15. The seed produced by the transgenic plant of claim 14.

16. A method for producing a transgenic plant, the method comprising:

providing a cell or tissue isolated from the transgenic plant tissue or plant of claim 8;

providing at lease one targeting endonuclease or a first nucleic acid molecule comprising a nucleic acid sequence encoding the at least one targeting endonuclease, wherein the at least one targeting endonuclease (s) recognize the at least one targeting endonuclease recognition site introduced into the genome of the transgenic plant;

providing a second nucleic acid molecule comprising a nucleic acid sequence of interest, and two additional nucleic acid sequences flanking the nucleic acid sequence of interest, wherein each of the two additional nucleic acid sequences is homologous with one of the two regions of nucleic acid sequence that lack sequence homology with genomic DNA;

introducing (i) the at least one targeting endonuclease or the first nucleic acid molecule; and

(ii) the second nucleic acid molecule into the plant cell or tissue, wherein the nucleic acid sequence of interest is stably integrated into the genome of the plant cell; and

regenerating a plant from the plant cell or tissue transformed with the first and second nucleic acid molecules.

17. The transgenic plant produced by the method of claim 16.

18. The seed produced by the transgenic plant of claim 17.

19. The method of claim 1, wherein the nucleic acid molecule is flanked on each end by one or more additional targeting endonuclease recognition sites.

20. The method of claim 19, further comprising introducing into the plant tissue or plant one or more targeting endonucleases that recognize the one or more additional targeting endonuclease recognition sites, wherein the nucleic acid molecule is excised from the genome of the plant tissue or plant.

21. The method according to claim 16, wherein the targeting endonuclease is a zinc finger nuclease.