A CRISPRi system for efficient and rapid gene knockdown in Caulobacter crescentus

CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knockdown gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus. Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically-dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter. We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in Caulobacter. RNA-sequencing experiments from Caulobacter crescentus CB15N cells expressing dCas9 and sgRNA targeting ctrA. (i) RNA-seq in ML3173 (Pxyl-dcas9(Sth3)) repressed in glucose, (ii) RNA-seq in ML3173 (Pxyl-dcas9(Sth3)) induced in xylose, (iii) RNA-seq in ML3174 (Pxyl-dcas9(Sth3) Pconstitutive-sgRNA(Sth3)_ctrA) repressed in glucose, and (iv) RNA-seq in ML3174 (Pxyl-dcas9(Sth3) Pconstitutive-sgRNA(Sth3)_ctrA) induced in xylose.

CRISPR干扰（CRISPR interference, CRISPRi）是一类可应用于多种生物体的新型高效工具，能够为基因表达敲低提供快速、特异且可靠的技术手段。新月柄杆菌（Caulobacter crescentus）是一种被广泛研究的模式细菌，尽管目前已开发出多种遗传操作工具，但通过该体系删除或耗竭单个基因仍需数周时间，这极大限制了相关遗传学研究的推进。本研究针对新月柄杆菌优化了CRISPRi技术方案，以实现其基因表达的特异性下调。尽管其他生物体中广泛使用的化脓链球菌（Streptococcus pyogenes）CRISPRi系统在新月柄杆菌中无法有效发挥功能，但本研究证实，源自嗜热链球菌（Streptococcus thermophilus）II型CRISPR3模块的催化失活型Cas9（dCas9），或是巴斯德链球菌（Streptococcus pasteurianus）来源的dCas9，均可在新月柄杆菌中实现高效应用。研究表明，上述CRISPRi系统可在异步或同步生长的细胞群体中，快速且可诱导地耗竭ctrA与gcrA这两种新月柄杆菌中已被深入研究的必需基因。此外，本研究还证实了基于CRISPRi的多基因敲低技术能力，为新月柄杆菌的系统性遗传互作研究开辟了全新方向。本研究包含表达dCas9与靶向ctrA的单向导RNA（single guide RNA, sgRNA）的新月柄杆菌CB15N菌株的RNA测序实验数据，具体涵盖以下四组数据：(i) 葡萄糖条件下阻遏表达的ML3173菌株（Pxyl-dcas9(Sth3)）RNA-seq数据；(ii) 木糖条件下诱导表达的ML3173菌株（Pxyl-dcas9(Sth3)）RNA-seq数据；(iii) 葡萄糖条件下阻遏表达的ML3174菌株（Pxyl-dcas9(Sth3) Pconstitutive-sgRNA(Sth3)_ctrA）RNA-seq数据；(iv) 木糖条件下诱导表达的ML3174菌株（Pxyl-dcas9(Sth3) Pconstitutive-sgRNA(Sth3)_ctrA）RNA-seq数据。