Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae . However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose‐fermenting, lactate‐producing industrial yeast strains, in which ALD6 , PHO13 , LEU2 , and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.