(629b) Genome Scale Loss-of-Function Screening in Saccharomyces Cerevisiae Using Crispr/Cas9 System

The baker’s yeast, Saccharomyces cerevisiae, represents a useful host for heterologous production of therapeutic proteins, biofuels and value-added chemicals. Resistance to harsh industrial fermentation conditions, such as low pH, high temperature or inhibitory products, is thus a desired trait. Despite the great effort in engineering high-performing yeast strains, only minimal success has been achieved, largely due to the low efficiency and throughput of yeast genome engineering. Previously, we developed a homology-integrated CRISPR/Cas (HI-CRISPR) system capable of knocking out single genes in Saccharomyces cerevisiae with near 100% efficiency. Here we sought to exploit this system for rapid, genome wide loss-of-function screening to improve complex yeast phenotypes. A total of 24765 homology integrated crRNAs were designed to target 6458 open reading frames (ORFs) including essential genes, with an average of 4 crRNAs targeting each ORF. The crRNA library was synthesized on chip, pooled and cloned into an ultrahigh copy number plasmid already expressing Cas9 and tracrRNA. A yeast mutant library with single gene disruption can be rapidly generated in approximately 6 days. Coupled with next generation sequencing, our method is suitable for both positive and negative screening. Furthermore, iterative rounds of genome wide mutagenesis can be conducted using the same plasmid library to enrich multiple mutations into a single cell, enabling the previously unfeasible screening of yeast mutant libraries with multiple knock-outs. We envision our method to be a useful tool for yeast metabolic engineering as well as understanding yeast genetics.