Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

Genome-wide CRISPR screens are a potent tool to identify the genetic underpinnings of engineered phenotypes. These screens have been extensively used, but identifying active guide RNAs is still a limitation. To overcome this limitation, we developed a strategy to allow the quantification of the cutting efficiency of each guide RNA in a synthesized library for the yeast Yarrowia lipolytica. Performing of the screen in the presence and absence of native nonhomologous DNA repair enabled the quantification of guide RNA activity for all guide RNAs. Identification of active guide RNAs improved essential gene classification, and the library was then used to evolve the industrially relevant phenotypes of high lipid accumulation and the elimination of pseudohyphal growth.