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Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.

  • Author(s): Schwartz, Cory
  • Cheng, Jan-Fang
  • Evans, Robert
  • Schwartz, Christopher A
  • Wagner, James M
  • Anglin, Scott
  • Beitz, Adam
  • Pan, Weihua
  • Lonardi, Stefano
  • Blenner, Mark
  • Alper, Hal S
  • Yoshikuni, Yasuo
  • Wheeldon, Ian
  • et al.
Abstract

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

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