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A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9
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A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9
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Journal Article
A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9
2018
Overview
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
.
CRISPR interference (CRISPRi) is a method for targeted silencing of transcription that requires the coexpression of protein dCas9 and a customized guide RNA. Here, Cui et al. show that certain guide RNAs induce toxicity in
E. coli
, and provide design rules to minimize off-target effects.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 38/23
/ 38/77
/ 49
/ 49/47
/ Biochemistry, Molecular Biology
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ CRISPR-Associated Protein 9 - chemistry
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Associated Protein 9 - metabolism
/ E coli
/ Escherichia coli - growth & development
/ Escherichia coli - metabolism
/ Fitness
/ Genomes
/ Genomics
/ Homology
/ Humanities and Social Sciences
/ RNA
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ RNA, Guide, CRISPR-Cas Systems - metabolism
/ Science
/ Screens
/ Toxicity
