High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Our results also highlight the fact that off-targets with as little as nine nucleotides of homology to the guide RNA can strongly block gene expression. Altogether this study provides important design rules to safely use dCas9 in E. coli.

No 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.

Bacteria from the same species can differ widely in their gene content. In E. coli, the set of genes shared by all strains, known as the core genome, represents about half the number of genes present in any strain. While recent advances in bacterial genomics have enabled to unravel genes required for fitness in various experimental conditions at the genome scale, most studies have focused on model strains. As a result, the impact of this genetic diversity on core processes of the bacterial cell largely remains to be investigated. Here, we developed a new CRISPR interference platform for highthroughput gene repression that is compatible with most E. coli isolates and closely-related species. We applied it to assess the importance of ~3,400 nearly ubiquitous genes in 3 growth media in 18 representative E. coli strains spanning most common phylogroups and lifestyles of the species. Our screens highlighted extensive variations in gene essentiality between strains and conditions. Unlike variations in gene expression level, variations in gene essentiality do not recapitulate the strains' phylogeny. Investigation of the genetic determinants for these variations highlighted the importance of epistatic interactions with mobile genetic elements. In particular, we showed how mobile genetic elements can trigger the essentiality of core genes that are usually nonessential. This study provides new insights into the evolvability of gene essentiality and argues for the importance of studying various isolates from the same species in bacterial genomics..

This study provides a link between stress and initiation of inflammatory attacks in patients with FMF. REDD1 emerges as a regulator of neutrophil function upstream to pyrin, is involved in NET release and regulation of IL-1β, and might constitute an important piece in the IL-1β-mediated inflammation puzzle.

The arms race between bacteria and phages led to the emergence of a variety of genetic systems used by bacteria to defend against viral infection, some of which were repurposed as powerful biotechnological tools. While numerous defense systems have been identified in genomic regions termed defense islands, it is believed that many more remain to be discovered. Here, we show that P2- like prophages and their P4-like satellites have genomic hotspots that represent a significant source of novel anti-phage systems. We validate the defense activity of 14 systems spanning various protein domains and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Immunity hotspots are present across prophages of distant bacterial species, highlighting their biological importance in the competition between bacteria and phages.

13High-throughput genetic screens are powerful methods to identify genes linked to a given 14 phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently 15 used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide 16 CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the 17 chromosome of E. coli. We first investigate the utility of this method for the prediction of essential 18 genes and various unusual features in the genome of E. coli. We then apply the screen to discover E.

Integral outer membrane proteins (OMPs) are crucial for the maintenance of the proteobacterial envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OM proteins (OMPs) activates the sigmaE (σE) transcriptional response. σE upregulates OMP-biogenesis factors, including the b-barrel assembly machinery (BAM) that catalyzes OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood OM lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating to OM-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the OM, thus supporting OMP biogenesis and OM integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.