We disclose a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a desired replacement sequence, 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 a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence flanked at its 5′ and 3′ sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence and a second DNA fragment comprising an expression cassette comprising a gene encoding a selection marker operably linked to regulatory sequences functional in the eukaryotic cell, transforming the modified eukaryotic cells with the DNA molecule, selecting transformed progeny cells having the DNA molecule inserted into the chromosome, deselecting transformed progeny cells having the DNA molecule inserted into the chromosome via NHR by expression of the selection marker, and obtaining cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence.