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

Metab Eng. 2019 Sep;55:102-110. doi: 10.1016/j.ymben.2019.06.007. Epub 2019 Jun 16.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems*
  • Gene Editing*
  • Gene Library*
  • Genes, Fungal*
  • Yarrowia / genetics*