Genome-wide CRISPR-dCas9 screens inE. coliidentify essential genes and phage host factors

Abstract

AbstractHigh-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the chromosome ofE. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome ofE. coli. We then apply the screen to discoverE. coligenes required by phages λ, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by λ enables to identify genes required for the formation of functional λ capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.