Methods and means for exact replacement of target DNA in eukaryotic organisms

摘要

Methods and means are provided for the exact exchange in eukaryotic cells, such as plant cells, of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print employing a method for the removal of a selected DNA flanked by two nucleotide sequences in direct repeats.

主权项

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
i. said DNA sequence of interest located between two flanking DNA regions of at least 10 nucleotides in length and having at least 80% sequence homology to a DNA region flanking said target DNA sequence, and preferably flanking said preselected site in the genome of said eukaryotic cell;ii. a selectable or screenable marker gene located between said flanking DNA regions, said selectable or screenable marker gene further being located between a first repeat sequence consisting of a 5′-terminal part of said preselected site and a second repeat sequence consisting of a 3′ terminal part of said preselected site, whereby the sequences common between said first and second repeat sequences are in direct repeat and are more than 14 nucleotides in length; andiii. at least one recognition site for a second DSBI enzyme located between said one of the flanking DNA regions and said first and second repeat sequence; 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.