CRISPR/Cas‐based screening of a gene activation library in Saccharomyces cerevisiae identifies a crucial role of OLE1 in thermotolerance

Summary CRISPR/Cas‐based (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) screening has been proved to be an efficient method to study functional genomics from yeast to human. In this study, we report the development of a focused CRISPR/Cas‐based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. The gene activation library was subjected to screening at 42°C, and the same library cultured at 30°C was set as a control group. After five successive subcultures, five clones were randomly picked from the libraries cultured at 30 and 42°C, respectively. The five clones selected at 30°C contain the specificity sequences of five different single guide RNAs, whereas all the five clones selected at 42°C contain the specificity sequence of one sgRNA that targets the promoter region of OLE1. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant. This study described the application of CRISPR/Cas‐based gene activation screening with an example of thermotolerant yeast screening, demonstrating that this method can be used to identify functional genes in yeast. In this study, we report the development of a focused CRISPR/Cas‐based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant.