A CRISPR Interference 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 knock down gene expression. 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 Although the CRISPRi system commonly used in other organisms does not work efficiently in , we demonstrate that a catalytically dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of or from can each be effectively used in We show that these CRISPRi systems can be used to rapidly and inducibly deplete or , two essential well-studied genes in , 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 is a major model organism for understanding cell cycle regulation and cellular asymmetry. The current genetic tools for deleting or silencing the expression of individual genes, particularly those essential for viability, are time-consuming and labor-intensive, which limits global genetic studies. Here, we optimized CRISPR interference (CRISPRi) for use in Using CRISPR3 or CRISPR systems, we show that the coexpression of a catalytically dead form of Cas9 (dCas9) with a single guide RNA (sgRNA) containing a seed region that targets the promoter region of a gene of interest efficiently downregulates the expression of the targeted gene. We also demonstrate that multiple sgRNAs can be produced in parallel to enable the facile silencing of multiple genes, opening the door to systematic genetic interaction studies. In sum, our work now provides a rapid, specific, and powerful new tool for silencing gene expression in and possibly other alphaproteobacteria.