The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. Other relevant data supporting the findings of the study are available in this article and its Supporting Information files.įunding: This work was supported by the European Research Council (ERC) under the Europe Union’s Horizon 2020 research and innovation program (grant agreement No ), by the French Government's Investissement d'Avenir program and by Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID), and by the Pasteur-Weizmann consortium F.R is supported by a doctoral scholarship from Ecole Normale Supérieure. The data from the essentiality screen can also be visualized on CRISPRbrowser, a web-based genome browser available at.
VIMR QUIT UNEXPECTEDLY CODE
The code and data tables used to perform the analyses are available at. The processed screen results are provided as supplementary tables.
VIMR QUIT UNEXPECTEDLY ARCHIVE
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: The sequencing data is available from the European Nucleotide Archive with the accession number PRJEB28256. Received: JAccepted: OctoPublished: November 7, 2018Ĭopyright: © 2018 Rousset et al. PLoS Genet 14(11):Įditor: Melanie Blokesch, Swiss Federal Institute of Technology Lausanne (EPFL), SWITZERLAND coli identify essential genes and phage host factors. Altogether, our results demonstrate the usefulness of genome-wide CRISPR-dCas9 screens in bacteria to uncover genes involved in various phenotypes.Ĭitation: Rousset F, Cui L, Siouve E, Becavin C, Depardieu F, Bikard D (2018) Genome-wide CRISPR-dCas9 screens in E. 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.
coli and for the production of functional progeny by phage λ.
We then apply our method to detect bacterial genes required by three different bacteriophages to kill E. 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. 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. 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. Over the past few years, CRISPR-Cas technologies have emerged as powerful tools to edit genomes and modulate gene expression. 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. 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. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. 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. Our data can be visualized on CRISPRbrowser, a custom web interface available at. 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. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential.
To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. 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.
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. High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype.