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

Robert W Bradley

doi:10.1016/j.btre.2021.e00680

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

Biotechnol Rep (Amst). 2021 Oct 9:32:e00680. doi: 10.1016/j.btre.2021.e00680. eCollection 2021 Dec.

Author

Robert W Bradley¹

Affiliation

¹ Department of Life Sciences, Imperial College London, SW2 5LU, United Kingdom.

Abstract

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.

Keywords: CRISPRi; Inducible repression; One-pot assembly.