ena-STUDY-INSTITUT PASTEUR-17-08-2018-10:40:08:241-1014
Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors
High-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 of E. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome of E. coli. We then apply the screen to discover E. coli genes required by phages ?, 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 ? enables to identify genes required for the formation of functional ? 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.
High-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 of E. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome of E. coli. We then apply the screen to discover E. coli genes required by phages ?, 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 ? enables to identify genes required for the formation of functional ? 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.