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 starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E . coli . To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E . coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr . We then apply the screen to discover E . coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics. Author summary Over the past few years, CRISPR-Cas technologies have emerged as powerful tools to edit genomes and modulate gene expression. They have been applied to perform high-throughput genetic screens with the purpose to understand the function of genes in a systematic manner, but the application of these screens to bacteria have so far remained limited. Here, we present the use of a library of ~92,000 guide RNAs directing the dCas9 protein to silence one by one all the genes in the chromosome of E . coli . To benchmark our method, we first investigate the performance of the technique to identify essential genes, highlighting several non-essential genes also found to be essential by other methods. We then apply our method to detect bacterial genes required by three different bacteriophages to kill E . coli and for the production of functional progeny by phage λ. Our screens highlight previously known and new genetic interactions between phages and their host’s pathways and emphasize the importance of bacterial capsule in the resistance to multiple phages. Altogether, our results demonstrate the usefulness of genome-wide CRISPR-dCas9 screens in bacteria to uncover genes involved in various phenotypes.
【초록키워드】 Gene Expression, Genome, Genetic, CRISPR, Screen, pathway, phenotype, sgRNA, bacteriophage, Bacteria, genomes, mutant, utility, dCas9, predict, mechanism, CRISPRi, Bacterial, Cas9, Interaction, Plasmid, bacteriophages, resistance mechanism, genetic interactions, genetic screens, bacterial capsule, Phenotypes, chromosome, technique, expression level, growth, medium, infection process, powerful tool, sgRNAs, library, convenience, random, phage, phages, bacterial genes, Cas9 RNA-guided nuclease, colanic acid, CRISPR-dCas9 screen, dCas9 protein, High-throughput genetic screens, phage λ, transduction assay, progeny, over, Host, Genes, Cell, highlight, guide RNA, tested, identify, detect, involved, reported, addition, remained, required, applied, functional, modulate, catalytic, highlighting, kill, bacterial gene, capsids, CRISPR-Ca, E . coli, High-throughput genetic screen, phages λ, 【제목키워드】 Factor, Host, identify,