Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors

Abstract

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.

A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

“…GuideFinder is also capable of designing guides for multiguide targeting, which may improve the efficacy of knockdown. Aside from the fact that overlapping guides have been shown to reduce knockdown efficiency, very little is known about the impact of the distance between dual targeting guides on gene knockdown in bacteria (6). However, it is plausible that the footprint of the Cas9 protein may influence the ability of two nearby guides to target simultaneously.…”

“…In the case of CRISPRi, dCas9 is used for transcriptional repression resulting from sterical blocking of transcription machinery and preventing initiation or elongation, depending on the location of the target sequence (on the promoter or DNA strand). CRISPRi has been recently applied on a genomewide scale to identify essential genes and phage host factors in Escherichia coli (6,7). Given the ease of CRISPRi, this technology has the power to investigate gene function in a variety of genetically diverse, nonmodel microorganisms on a genomewide scale; thus, flexible programs for high-throughput guide design in draft bacterial are critically needed.…”

Impact of CRISPR interference on strain development in biotechnology

“…This technology becomes more attractive when combined with high‐throughput sequencing and dedicated bioinformatics analysis [56,57]. As an example, a genome‐wide CRISPRi screen with a starting pool of 92,000 sgRNAs that targeted random positions in the E. coli chromosome was used to determine gene essentiality during the growth of this bacterium [58]. This approach confirmed gene essentiality for 79% of previously reported genes and revealed that a number of genes previously annotated as essential are actually not essential.…”

“…In addition, the screen was applied to discover E. coli genes required by phages λ, T4, and 186 to kill their host. Several host pathways were important in the infection processes of these phages, with the biosynthesis of colanic acid being relevant for resistance to all three phages [58]. In functional genomics of the rapidly replicating bacterium Vibrio natriegens with CRISPRi, functionally relevant genes were identified with a gRNA library targeting 4,565 (99.7%) protein‐coding genes [59].…”

Gene silencing with CRISPRi in bacteria and optimization of dCas9 expression levels

“…This phenomenon called the "bad-seed" effect is particularly observed at high dCas9 concentrations, and could be decreased by tuning dCas9 levels while maintaining strong on-target repression. Using such optimized expression cassette enables performing powerful screens using dCas9 [28].…”