Insertion orientation within the cassette affects gene-targeting success during ends-out recombination in the yeast Saccharomyces cerevisiae.

Abstract

Gene-targeting is one of the most important molecular tools for genomic manipulations for research and industrial purposes. However, many factors influence targeting fidelity undermining the efforts for accurate, fast, and reliable construction of genetically modified yeast strains. Therefore, it is of great academic interest that we uncover as many as possible parameters affecting the recombination mechanisms that enable targeting. Since usually, researchers choose the orientation of the insertion (marker) within the module at random, it seemed interesting to see whether the same module will achieve essentially the same targeting efficiency when the same marker was oriented alternatively concerning the same target gene. Thus, two loci (URA3 and LEU2) and one allele (ura3-52) in a haploid yeast genetic background were targeted by artificial modules bearing homologous insertions in alternative orientations being flanked by long asymmetrical targeting homology to either replace or disrupt a genomic target. Results showed that insertion orientation within the targeting module strongly influences targeting in yeast, regardless of the targeting approach.