Patent ID: 8367890

Claim:
A method for exchanging a target DNA sequence in the genome of a plant for a DNA sequence of interest comprising a. inducing a double stranded DNA break at a preselected site in the genome of a cell of a plant, said preselected site being located within said target DNA sequence or in the vicinity of said target DNA sequence; b. introducing a repair DNA molecule of interest into said plant cell, said DNA molecule comprising i. said DNA sequence of interest located between two flanking DNA regions being at least 10 nucleotides in length and having at least 80% sequence homology to a DNA region flanking said target DNA sequence; ii. a selectable or screenable marker gene located between said flanking DNA regions, said selectable or screenable marker gene further being located between one of said flanking DNA regions and a copy of at least part of said one of said flanking DNA regions in direct repeat orientation; iii. at least one recognition site for a DSBI enzyme located between said one of said flanking DNA regions and said copy of at least part of said flanking DNA region; c. selecting a population of plant cells comprising said selectable or screenable marker; d. selecting a plant cell wherein said selectable or screenable marker has been introduced by homologous recombination through said flanking DNA regions and regenerating a plant from said plant cell; e. crossing said regenerated plant or a progeny plant thereof comprising said selectable marker gene with a plant comprising a chimeric gene encoding a DSBI enzyme recognizing said recognition site located in said repair DNA molecule of interest to obtain a population of F1 progeny plants, said chimeric gene comprising the following operably linked DNA segments: i. a germline specific promoter, provided that said promoter does not comprise the nucleotide sequence of SEQ ID No 3 from position 1-992; ii. a DNA region encoding said DSBI enzyme recognizing said recognition site located in said repair DNA molecule of interest; iii. a transcription termination and polyadenylation region; f. selecting an F1 progeny plant comprising said selectable or screenable marker gene and said DSBI enzyme encoding chimeric gene; g. crossing said progeny plant with a wild type plant to obtain a population of F2 progeny plants; h. selecting a population of F2 progeny plants which comprises said DSBI enzyme encoding chimeric gene; and i. selecting an F2 progeny plant wherein said selectable or screenable marker gene is deleted by homologous recombination between said one of the flanking DNA regions and a partial flanking DNA region comprising part of said one of the flanking DNA regions.