A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9 - Institut Pasteur Access content directly
Journal Articles Nature Communications Year : 2018

A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9

Abstract

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.
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pasteur-01819630 , version 1 (20-06-2018)

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Lun Cui, Antoine Vigouroux, François Rousset, Hugo Varet, Varun Khanna, et al.. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nature Communications, 2018, 9 (1), pp.1912. ⟨10.1038/s41467-018-04209-5⟩. ⟨pasteur-01819630⟩
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