Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a.

Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16 bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.