EXCISION OF TRANSGENES IN GENETICALLY MODIFIED ORGANISMS

• US 20110191877A1
• 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: Petolino, Joseph F., Russell, Sean M.
• Granted Patent: US 9,695,432 B2
• Time in Patent Office: Days
• US Class Current: 800/260
• 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
• CPC Class Codes: C12N 15/8265 Transgene containment, e.g. gene dispersal
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

A method for deleting a region of DNA in a plant. In some embodiments, the method comprises transforming a plant with a nucleic acid molecule, wherein the nucleic acid molecule encodes one or more zinc finger nuclease(s) (ZFNs) operably linked to one or more tissue-specific promoter(s), e.g., a pollen-specific promoter. Methods include excising native genes in a plant. Accordingly, in some embodiments, ZFNs are engineered that recognize sequences that flank native plant genes. In further embodiments, ZFNs are expressed under the control of developmental stage-specific promoters, such that, for example, nucleic acid sequences are specifically excised in plants during relatively late stages of development. Nucleic acid molecules useful for carrying out disclosed methods and plants produced by the methods are included.

Claims

1. A method for deleting a region of DNA in a plant, the method comprising:

providing a viable plant containing a genomic DNA, the genomic DNA comprising the region of DNA; and

introducing or expressing a zinc finger nuclease in the viable plant containing the genomic DNA, wherein the zinc finger nuclease is engineered to cleave the genomic DNA at a recognition sequence;

wherein the method results in cleavage of the genomic DNA at recognition sequences and in the excision of the genomic DNA, wherein the region of DNA is absent from the genomic DNA.

2. The transgenic plant produced by the method of claim 1.

9. A method of producing a transgenic plant comprising:

transforming a plant cell or plant tissue with the isolated nucleic acid molecule of claim 1; and

regenerating a whole plant.

10. The transgenic plant produced by the method of claim 9.

15. A method for reducing the transmission of a gene of interest to other plants comprising:

crossing the plant of claim 9 with a plant regenerated from a plant cell or tissue transformed with an isolated nucleic acid molecule comprising a pollen-specific promoter operably linked to a zinc finger nuclease, wherein the gene of interest is specifically excised in pollen of the progeny resulting from the cross and the native gene of interest is excised; and

cultivating the progeny resulting from the cross.

3. A method for deleting a region of DNA in a plant, the method comprising:

providing a first viable plant containing a genomic DNA, the genomic DNA comprising the region of DNA and a first recognition sequence flanking the 3′

end and a second recognition sequence flanking the 5′

end of the region of DNA;

providing a second viable plant containing a genomic DNA, the genomic DNA comprising a DNA encoding a zinc finger nuclease engineered to cleave the genomic DNA at the recognition sequences;

crossing the first and second viable plants such that F1 seed is produced on either the first or the second viable plant; and

growing a resultant F1 plant containing a genomic DNA, wherein the region of DNA is absent from the genomic DNA.

4. The method of claim 3, wherein the first recognition sequence and the second recognition sequence are identical.

5. The transgenic plant produced by the method of claim 3.

6. An isolated nucleic acid molecule comprising:

a first nucleic acid sequence recognized by a zinc finger nuclease;

a gene of interest; and

a second nucleic acid sequence recognized by a zinc finger nuclease, wherein the gene of interest is flanked by the first and second nucleic acid sequences recognized by a zinc finger nuclease.

7. The isolated nucleic acid molecule of claim 6, further comprising a promoter operably linked to the gene of interest.

8. The isolated nucleic acid molecule of claim 6, wherein the first nucleic acid sequence and the second nucleic acid sequence are flanked by homologous sequences.

11. An isolated nucleic acid molecule comprising:

a promoter; and

a nucleic acid sequence encoding a zinc finger nuclease, wherein the promoter is operably linked to the nucleic acid sequence encoding the zinc finger nuclease, wherein the nucleic acid sequence is flanked by zinc finger nuclease cleavage sites.

12. A method of producing a transgenic plant comprising:

transforming a plant cell or plant tissue with the isolated nucleic acid molecule of claim 11; and

regenerating a whole plant.

13. The isolated nucleic acid molecule of claim 11, wherein the promoter is selected from the group consisting of:

a pollen-specific promoter, a seed-specific promoter, and a developmental-stage specific promoter.

14. A method of excising a gene of interest in a plant comprising:

crossing the plant of claim 9 with the plant of claim 11, wherein the progeny resulting from the cross exhibit zinc finger nuclease-mediated excision of the gene of interest.

16. A method of excising a native gene of interest in a plant comprising:

transforming a plant cell or tissue comprising a gene of interest with an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a zinc finger nuclease or an isolated protein sequence which encodes a zinc finger nuclease, wherein the zinc finger nuclease recognizes a nucleic acid sequence flanking the native gene of interest; and

regenerating a whole plant.

17. A method for deleting a region of DNA in a plant containing a nucleic acid molecule comprising:

providing a first nucleic acid sequence recognized by a zinc finger nuclease;

providing a selectable marker gene expression cassette; and

providing a second nucleic acid sequence recognized by a zinc finger nuclease, wherein the selectable marker is flanked by the first and second nucleic acid sequences recognized by a zinc finger nuclease.

18. The method of claim 17, wherein the first nucleic acid sequence and the second nucleic acid sequence are flanked by homologous sequences.

19. The method of claim 17, wherein a zinc finger nuclease engineered to cleave genomic DNA at a recognition sequence is expressed or introduced in a viable plant cell, thereby resulting in cleavage of the genomic DNA at recognition sequences resulting in the excision of the genomic DNA, wherein the selectable marker is absent from the genomic DNA.

20. The method of claim 17, wherein each half of a zinc finger nuclease monomer is expressed separately and when paired in conjunction with one another form a functional complex.