Unexpectedly toxicity of dCas9 gRNAs in E. coli is due to very short sequence off-target effets on essential genes.. Sequence-specific toxicity of dCas9 in bacteria

The catalytic null mutant of Cas9 (dCas9) is a powerful tool to silence gene expression in bacteria. Pooled CRISPR-dCas9 screens can be used to investigate the effect of individual genes in high-throughput and have been used to study essential genes or decipher drugs mode of action. Recently, a screen performed in our lab revealed that many guide RNAs (sgRNA) produce an unexpectedly strong fitness defect in E. coli, an effect determined by the five PAM-proximal bases (seed) of the guide. Here, we investigated the molecular mechanism and cellular consequences of this bad seed effect using a combination of RNA-seq, ChIP-Seq, high-throughput screening and genetics approaches. We demonstrated that dCas9 binding to off-targets with as little as four nucleotides of complementarity to the sgRNA can block the expression of essential genes, resulting in drastic effects on E. coli physiology. We further explored how the off-target sequence and context influence the bad seed effect, and compare bad seeds among diverse E. coli and gammaproteobacterial isolates. Altogether, our results will help improve future applications of this extensively used biotechnological tool.