An easy-to-use CRISPRi plasmid tool for inducible knockdown in E. coli

•An easy-to-use plasmid tool for CRISPRi transcription repression in bacteria.•Dual inducible promoters for tight control of CRISPRi components.•One-day cloning protocol to insert targeting spacer sequences.•Strong repression of plasmid-borne and chromosomal targets. CRISPR-interference (CRISPRi) is a powerful genetic tool with wide application in bacteria. In this work a single plasmid system is presented which expresses deactivated Cas9 and a short guide RNA sequence from separate inducible promoters, enabling up to 40-fold repression of target genes with minimal leaky repression in the uninduced state. The plasmid is designed for rapid spacer insertion with easy screening for correct clones, and a simple one-day protocol with >99.9% assembly efficiency is described. This plasmid is made available to the research community with a view to facilitating wider use of CRISPRi in bacterial systems.