Engineered landing pads for gene targeting in plants

Patent metadata

Field	Value
Publication number	US-10160975-B2
Application number	US-201414458086-A
Country	US
Kind code	B2
Filing date	Aug 12, 2014
Priority date	Jan 22, 2010
Publication date	Dec 25, 2018
Grant date	Dec 25, 2018

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.

First claim

What is claimed is:

1. A method for producing a transgenic plant cell, the method comprising:
transforming a plant cell with a nucleic acid molecule comprising a polynucleotide that comprises at least two nucleotide sequences selected from the group consisting of SEQ ID NOs:11-14, 17, and 18, and at least one targeting endonuclease recognition site, wherein the at least two nucleotide sequences selected from the group consisting of SEQ ID NOs:11-14, 17, and 18 flank the at least one targeting endonuclease recognition site, such that the polynucleotide is stably integrated into the genome of the plant cell.

2. The method according to claim 1 , wherein the polynucleotide further comprises non-identical restriction sites.

3. The method according to claim 2 , wherein the non-identical restriction sites comprise compatible single-stranded ends that allow concatemerization of multiple nucleic acids that each comprise:
at least two regions of nucleic acid sequence that are not identical to a DNA sequence from the genome of a plant cell, and at least one targeting endonuclease recognition site, wherein the at least two regions of nucleic acid sequence that are not identical to any genomic DNA of the plant cell flank the at least one targeting endonuclease recognition site.

4. The method of claim 1 , wherein the nucleic acid molecule comprises a polynucleotide selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:19.

5. The method of claim 1 , wherein the nucleic acid molecule is a plant transformation vector.

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

7. The method according to claim 1 , wherein the polynucleotide is stably integrated randomly in the genome of the plant cell.

8. The method according to claim 1 , wherein the polynucleotide is stably integrated at one or more known target sites in the genome of the plant cell.

9. The method of claim 1 , wherein the polynucleotide is stably integrated into an amplified region of a chromosome or a minichromosome.

10. The transgenic plant cell produced by the method according to claim 1 .

11. A method for modifying the genome of a transgenic plant cell, the method comprising introducing into the plant cell of claim 10 :
at least one targeting endonuclease that recognizes the targeting endonuclease recognition site in the polynucleotide; and
a nucleic acid molecule comprising a nucleotide sequence of interest and two additional nucleotide sequences flanking the nucleotide sequence of interest, wherein each of the two additional nucleotide sequences is homologous with one of the nucleotide sequences in the polynucleotide selected from the group consisting of SEQ ID NOs: 11-14, 17, and 18,
such that the nucleotide sequence of interest is stably integrated into the genome of the plant cell.

12. The transgenic plant cell produced by the method according to claim 11 .

13. The method according to claim 11 , wherein the targeting endonuclease is a zinc finger nuclease.

14. The method according to claim 11 , wherein the targeting endonuclease is introduced into the plant cell by transforming the plant cell with a nucleic acid molecule encoding the targeting endonuclease.

15. The method according to claim 11 , the method further comprising regenerating a plant tissue from the transgenic plant cell.

16. The method according to claim 11 , the method further comprising regenerating a plant from the transgenic plant cell.

17. A plant produced by the method according to claim 16 , wherein the plant comprises the nucleotide sequence of interest.

18. A seed produced by the transgenic plant produced by the plant of claim 17 , wherein the seed comprises the nucleotide sequence of interest.

19. The method according to claim 1 , the method further comprising regenerating a plant tissue from the transgenic plant cell.

20. The method according to claim 1 , the method further comprising regenerating a plant from the transgenic plant cell.

21. A plant produced by the method according to claim 20 , wherein the plant comprises the polynucleotide.

22. A seed produced by the transgenic plant produced by the plant of claim 21 , wherein the seed comprises the polynucleotide.

23. The method according to claim 1 , wherein the polynucleotide further comprises one or more additional targeting endonuclease recognition sites that flank the at least two nucleotide sequences selected from the group consisting of SEQ ID NOs:11-14, 17, and 18 on each end of the polynucleotide.

24. The method according to claim 23 , the method further comprising regenerating a plant tissue or plant from the transgenic plant cell.

25. The method according to claim 24 , 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, such that the polynucleotide is excised from the genome of the plant tissue or plant.

Assignees

• Dow Agrosciences Llc

Inventors

• Ainley W Michael
• Blue Ryan C
• Murray Michael G
• Corbin David Richard
• Miles Rebecca Ruth
• Webb Steven R

Classifications

• C12N15/8213

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

• C12N15/8241

  Phenotypically and genetically modified plants via recombinant DNA technology · CPC title

• C12N15/8201 Primary

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title