Excision of transgenes in genetically modified organisms

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.

What is claimed is: 
     
         1 . A method for segregating a first polynucleotide fragment from a second polynucleotide fragment within a genome of a plant cell, wherein the first polynucleotide fragment and the second polynucleotide fragment are located in close proximity on a single chromosome, the method comprising the steps of:
 (a) providing the plant cell comprising the first polynucleotide fragment and the second polynucleotide fragment located in close proximity on the single chromosome, the plant cell further comprising at least a second chromosome, wherein the first and second chromosomes are homologous or homeologous chromosomes of each other;   (b) introducing a site specific nuclease into the genome of the plant cell;   (c) producing a double strand break in the first chromosome, wherein the double strand break in the first chromosome is introduced between the first polynucleotide fragment and the second polynucleotide fragment;   (d) producing a double strand break in the second chromosome, wherein the double strand break in the second chromosome is introduced between the first polynucleotide fragment and the second polynucleotide fragment; and   (e) obtaining a progeny plant comprising a modified genome, wherein the first polynucleotide fragment and the second polynucleotide fragment segregate from one another.   
     
     
         2 . The method of  claim 1 , wherein the site specific nuclease comprises a zinc finger nuclease, a TALEN nuclease, a CRISPR-Cas9 nuclease, a meganuclease, or a leucine zipper nuclease. 
     
     
         3 . The method of  claim 1 , wherein the site specific nuclease is delivered to the plant cell by intra-genomic recombination or via direct delivery. 
     
     
         4 . A plant produced by the method of  claim 1 . 
     
     
         5 . The plant of  claim 4 , wherein the plant is a transgenic plant. 
     
     
         6 . A plant part, fruit or seed obtained from the plant of  claim 4 . 
     
     
         7 . A method to segregate a first polynucleotide fragment from a second polynucleotide fragment within the genome of a progeny plant, the method comprising the steps of:
 (a) providing a plant cell comprising the first polynucleotide fragment and the second polynucleotide fragment located in close proximity on a single chromosome, the plant cell further comprising at least a second chromosome, wherein the chromosomes are homologous or homeologous chromosomes of each other;   (b) creating a double strand break in the first and second chromosome, wherein the double strand break in the homologous or homeologous chromosomes is introduced between the first polynucleotide fragment and the second polynucleotide fragment;   (c) segregating the first polynucleotide fragment from the second polynucleotide fragment located in close proximity on the single chromosome of the plant cell; and   (d) producing a progeny plant that does not contain the first polynucleotide fragment within the genome of the progeny plant.   
     
     
         8 . The method of  claim 7 , wherein the double strand break is produced by a site specific nuclease. 
     
     
         9 . The method of  claim 8 , wherein the site specific nuclease comprises a zinc finger nuclease, a TALEN nuclease, a CRISPR-Cas9 nuclease, a meganuclease, or a leucine zipper nuclease. 
     
     
         10 . The method of  claim 8 , wherein the site specific nuclease is delivered to the plant cell by intra-genomic recombination or via direct delivery. 
     
     
         11 . A plant produced by the method of  claim 7 . 
     
     
         12 . The plant of  claim 11 , wherein the plant is a transgenic plant. 
     
     
         13 . A plant part, fruit or seed obtained from the plant of  claim 11 . 
     
     
         14 . A method for segregating the inheritance of a first transgene from a second transgene in a progeny plant, the method comprising the steps of:
 (a) crossing a first parent plant with a second parent plant;   (b) producing a double strand break between the first transgene and the second transgene; and   (c) obtaining the progeny plant.   
     
     
         15 . The method of  claim 14 , further comprising screening the progeny plant for segregation of the first transgene from the second transgene in the progeny plant. 
     
     
         16 . The method of  claim 14 , wherein the double strand break is produced by a site specific nuclease. 
     
     
         17 . The method of  claim 16 , wherein the site specific nuclease comprises a zinc finger nuclease, a TALEN nuclease, a CRISPR-Cas9 nuclease, a meganuclease, or a leucine zipper nuclease. 
     
     
         18 . The method of  claim 16 , wherein the site specific nuclease is delivered to the plant by intra-genomic recombination or via direct delivery. 
     
     
         19 . A plant produced by the method of  claim 14 . 
     
     
         20 . The plant of  claim 19 , wherein the plant is a transgenic plant. 
     
     
         21 . A plant part, fruit or seed obtained from the plant of  claim 19 .