Base editor enables rational genome-scale functional screening for enhanced industrial phenotypes in Corynebacterium glutamicum

作者：Ye Liu, Ruoyu Wang, Jiahui Liu, Hui Lu, Haoran Li, Yu Wang, Xiaomeng Ni, Junwei Li, Yanmei Guo, Hongwu Ma, Xiaoping Liao, Meng Wang · 发表于：Science Advances · 年份：2022 · DOI：10.1126/sciadv.abq2157 · 被引用次数：23 · 研究领域：CRISPR and Genetic Engineering、Microbial Metabolic Engineering and Bioproduction、Viral Infectious Diseases and Gene Expression in Insects

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 Corynebacterium glutamicum 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, purU and serA were proven to be related to the furfural tolerance in C. glutamicum . 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.