Method for homologous recombination

Abstract

The present invention discloses a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a replacement sequence of interest comprising: modifying a parent eukaryotic cell with a preference for NHR to provide a eukaryotic cell having an increased HR/NHR ratio as compared to the parent cell, providing two sets of DNA molecules of which the first set comprises DNA molecules each comprising a first non-functional fragment of the replacement sequence of interest flanked at its 5′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence and the second set comprises DNA molecules each comprising a second non-functional fragment of the DNA replacement sequence of interest overlapping with the first non-functional fragment and flanked at its 3′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence, wherein the first and second non-functional fragments become functional upon recombination; transforming the modified eukaryotic cells with both sets of DNA molecules; and growing the cells to obtain transformed progeny cells having a DNA molecule from the first set recombined with a DNA molecule from the second set into a DNA molecule comprising a functional replacement sequence and having the target sequence replaced by the functional replacement sequence.

Dependencies are inferred from the claim text.