Format

Send to

Choose Destination
PLoS Genet. 2018 Nov 7;14(11):e1007749. doi: 10.1371/journal.pgen.1007749. eCollection 2018 Nov.

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

Author information

1
Synthetic Biology Group, Microbiology Department, Institut Pasteur, Paris, France.
2
Sorbonne Université, Collège Doctoral, Paris, France.
3
Hub Bioinformatique et Biostatistique, Institut Pasteur - C3BI, USR 3756 IP CNRS, Paris, France.

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.

PMID:
30403660
PMCID:
PMC6242692
DOI:
10.1371/journal.pgen.1007749
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center