Base editor enables rational genome-scale functional screening for enhanced industrial phenotypes in <i>Corynebacterium glutamicum</i>.

Abstract

Genome-scale functional screening accelerates comprehensive assessment of gene function in cells. Here, we have established a genome-scale loss-of-function screening strategy that combined a cytosine base editor with approximately 12,000 parallel sgRNAs targeting 98.1% of total genes in <i>Corynebacterium glutamicum</i> ATCC 13032. Unlike previous data processing methods developed in yeast or mammalian cells, we developed a new data processing procedure to locate candidate genes by statistical sgRNA enrichment analysis. Known and novel functional genes related to 5-fluorouracil resistance, 5-fluoroorotate resistance, oxidative stress tolerance, or furfural tolerance have been identified. In particular, <i>purU</i> and <i>serA</i> were proven to be related to the furfural tolerance in <i>C. glutamicum</i>. A cloud platform named FSsgRNA-Analyzer was provided to accelerate sequencing data processing for CRISPR-based functional screening. Our method would be broadly useful to functional genomics study and strain engineering in other microorganisms.