LAUSR

ABSTRACT For Saccharomyces cerevisiae, gene knockout is routinely performed by transformation with a linear DNA cassette consisting of a selection marker gene flanked by upstream and downstream sequences homologous to a target gene. Over the years, several plasmid sets containing a variety of selection marker genes have been developed. Targeting fidelity under this strategy was high when performing the first gene knockout in a strain. However, we found that targeting fidelity decreased substantially when performing subsequent gene knockouts. The majority of the transformants were “incorrect,” in which the new selection marker gene replaced a pre-existing selection marker gene instead of its intended target. This was caused by the presence of shared regions in the knockout DNA cassettes. To minimize shared regions among knockout cassettes, we developed a set of template plasmids, in which each selection marker open reading frame is flanked by a unique promoter/terminator combination. Our SJZ series templates cover eight selection markers, namely, URA3 (C. a.), TRP1 (K.l.), his5 (S.p.), LEU2 (K.l.), nat, hph, kan, and amdS. When using our templates, targeting fidelity in subsequent gene knockouts was restored to as high as that of the first knockout, with essentially all the transformants being correct. Our templates can therefore bring efficiency improvements in future research projects involving multi-gene knockouts. IMPORTANCE When knocking out multiple genes in yeast, recombination among selection markers produces a large portion of false-positive transformants. We developed a new set of templates designed to minimize shared regions among selection markers. The use of this new template set resulted in essentially all transformants being correct knockouts. When knocking out multiple genes in yeast, recombination among selection markers produces a large portion of false-positive transformants. We developed a new set of templates designed to minimize shared regions among selection markers. The use of this new template set resulted in essentially all transformants being correct knockouts.