(563b) Application and Validation of a Genome-Wide CRISPR-Cas9 Library for the Oleaginous Yeast Yarrowia Lipolytica
To achieve genome-wide knockout screening, we designed approximately 48,000 sgRNAs that targeted each gene with 6-fold coverage. The sgRNAs were designed to (i) maximize on-target cutting efficiency, (ii) target in the first 20% of each geneâs coding sequence, (iii) avoid targeting intronic sequences, and (iv) avoid targeting more than once within the genome. With the help of DOEâs Joint Genome Institute, we compared four different cloning strategies and constructed a pooled library that contained over 99% of the designed sgRNAs. In order to determine the efficacy of our library design and quantify the effectiveness of the sgRNAs, we transformed the pooled library into a strain of Y. lipolytica that expressed Cas9 and was deficient in DNA repair. In this strain, a double-stranded break in genomic DNA leads to cell death, meaning efficient sgRNAs are depleted and poor cutting sgRNAs are enriched with extended culture time. Isolation and sequencing of the plasmid library after several days of outgrowth enabled us to identify and rank the cutting efficiency of each designed sgRNA. We found that approximately 63% of the designed sgRNAs were highly efficient cutters, and that over 97% of the genes in the Y. lipolytica genome were targeted by at least one highly efficient sgRNA. The library was then transformed into a strain of Y. lipolytica expressing Cas9 and competent for DNA repair, so that a double-strand break in genomic DNA lead to an indel mutation to inactivate the targeted gene. By quantifying only the most efficient sgRNA targeting each gene, we identified approximately 1,500 genes that had at least a 10-fold reduction in sgRNA abundance. This work is one of the first examples of genome-wide engineering of a nonconventional yeast and provides a blueprint for how genome-wide CRISPR screens can be effectively implemented in other organisms.