Customized Crispr-Cas System for Multiple Simultaneous Gene Disruptions in Saccharomyces Cerevisiae

Efficient and convenient multiple simultaneous gene disruptions in model organism Saccharomyces cerevisiae holds great potential for both basic research and industrial applications. Current multiple simultaneous gene disruption strategies involve selection or screening, which are labor-intensive, time-consuming and can cause yeast genome instability. Here we customized the recently developed type II clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system for highly efficient and convenient multiple simultaneous gene disruptions in Saccharomyces cerevisiae. A single CRISPR-Cas expression cluster was constructed in a high copy number plasmid. After genome cleavage by Cas9, the target gene was disrupted through homologous recombination mediated double strand break repair by a mutagenizing dsDNA donor. The donor was synthesized together with the 20 base pair guide sequence for easy plasmid construction. Multiple donor and spacer pairs could be constructed in a single CRISPR array to perform one step multiple gene disruptions. Using this customized CRISPR-Cas system, three genes can1, ade2 and lyp1 were simultaneously disrupted in 4 days with an efficiency of 83%. The efficiency further reached to 93% after 8 days, which is high enough to skip selection or screening for multiple simultaneous gene disruptions, thus greatly reducing time and labor. The desired disruption strain can be easily obtained by random genotyping. This customized CRISPR-Cas genome editing tool represents an effective strategy for creating yeast strains with interesting or useful properties.