EXCISION OF TRANSGENES IN GENETICALLY MODIFIED ORGANISMS
First Claim
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.
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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.
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Citations
20 Claims
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1. A method for deleting a region of DNA in a plant, the method comprising:
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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. - View Dependent Claims (2, 9, 10, 15)
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3. A method for deleting a region of DNA in a plant, the method comprising:
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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. - View Dependent Claims (4, 5)
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6. An isolated nucleic acid molecule comprising:
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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. - View Dependent Claims (7, 8)
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11. An isolated nucleic acid molecule comprising:
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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. - View Dependent Claims (12, 13, 14)
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16. A method of excising a native gene of interest in a plant comprising:
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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.
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17. A method for deleting a region of DNA in a plant containing a nucleic acid molecule comprising:
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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. - View Dependent Claims (18, 19, 20)
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Specification