Advances and applications of CRISPR/Cas-mediated interference in Escherichia coli

The bacterium Escherichia coli (E. coli) is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds. In the past decade, the metabolic engineering of E. coli has undergone significant advances, although further productivity improvements will require extensive genome modification, multi-dimensional regulation, and multiple metabolic-pathway coordination. In this context, clustered regularly interspaced short palindromic repeats (CRISPR), along with CRISPR-associated protein (Cas) and its inactive variant (dCas), have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E. coli. In this review, we briefly describe the CRISPR/Cas9 technology in E. coli, and then summarize the recent advances in CRISPR/dCas9 interference (CRISPRi) systems in E. coli, particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing. Moreover, we discuss recent applications of the CRISPRi system for enhancing metabolite production in E. coli, and finally highlight the major challenges and future perspectives of this technology.