Patent ID: 8765448
Filing Date: 2014-07-01
Classification: C12N

Abstract:
1. A method for exchanging a target DNA sequence in the genome of a eukaryotic cell or eukaryotic organism for a DNA sequence of interest comprising the following steps: a. inducing a first double stranded DNA break at a preselected site in the genome of a cell of said eukaryotic organism, said preselected site being located within said target DNA sequence or within 1 kb of said target DNA sequence and said preselected site being recognized by a first double-stranded break inducing (DSBI) enzyme; b. introducing a repair DNA molecule into said eukaryotic cell, said repair DNA molecule comprising c. selecting a population of cells comprising said selectable or screenable marker; d. selecting a cell wherein said selectable or screenable marker has been introduced by homologous recombination through said flanking DNA regions; e. introducing a double stranded break at the recognition site for said second DSBI enzyme in said cell; and f. selecting a progeny cell wherein said selectable or screenable marker gene is deleted by homologous recombination between said direct repeats thereby recreating said preselected site.