Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity [RNA-Seq]. Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity [RNA-Seq]

Over the last decade, gene-silencing mediated by dCas9 binding to transcribed regions or their promoters, a strategy referred to as CRISPRi, has emerged as a powerful tool in bacterial genetics. While this strategy has already been broadly adopted, several studies have reported experimental setups in which dCas9 expression was toxic. In particular, guide RNAs that share specific PAM-proximal sequence motifs were shown to be toxic to E. coli. Here we demonstrate that this toxicity is caused by off-target binding of dCas9 to the promoter of essential genes. Silencing of off-target genes can occur with as little as 4nt of identity between the PAM-proximal sequence and the off-target position. This phenomenon only occurs in some promoter sequences but does not appear to be constrained to any specific PAM-proximal sequence. Accordingly, screens performed in various strains of E. coli and related species shows that the nature of toxic guide RNAs changes together with the evolution of the sequence of off-target positions. These results highlight the importance of relying on several guide RNAs targeting the same gene when performing CRISPRi experiments in bacteria in order to avoid any possible confounding results due to off-target binding. Overall design: Samples were prepared from E. coli LC-E18 harbouring psgRNAcos plasmids with 3 different guides (control guide, AGGAA_non1 and ACCCA_non1). Samples were harvested 3 hours after aTc induction.

过去十年间，由失活Cas9（dead Cas9, dCas9）结合转录区域或其启动子介导的基因沉默策略——即CRISPR干扰（CRISPR interference, CRISPRi）——已成为细菌遗传学领域的强力研究工具。尽管该策略已得到广泛应用，但多项研究报道称，dCas9的表达会产生细胞毒性。具体而言，携带特定PAM近端序列基序的向导RNA（guide RNA, gRNA）被证实会对大肠杆菌（Escherichia coli, E. coli）产生毒性。本研究证实，该毒性源于dCas9与必需基因启动子的脱靶结合（off-target binding）。脱靶基因的沉默仅需PAM近端序列与脱靶位点之间存在至少4个核苷酸（nucleotide, nt）的序列同源性。该现象仅发生于部分启动子序列中，且未局限于某一特定的PAM近端序列。因此，在不同大肠杆菌菌株及相关物种中开展的筛选实验显示，毒性向导RNA的特征会随脱靶位点序列的演化而发生改变。本研究结果凸显了在细菌中开展CRISPRi实验时，使用多条靶向同一基因的向导RNA的重要性，以规避脱靶结合可能导致的混杂性实验结果。实验整体设计：以携带含3种向导RNA（对照向导RNA、AGGAA_non1及ACCCA_non1）的psgRNAcos质粒的大肠杆菌LC-E18菌株制备样本，于aTc诱导3小时后收集样本。