Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,r...Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.展开更多
基金supported by the National Key Research and Development Program of China (2018YFA0901500)the National Natural Science Foundation of China (U2032210)。
文摘Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.
