A robust CRISPR interference gene repression system in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a significant cause of seafood-associated gastroenteritis and pestilence in aquaculture worldwide. Despite extensive research, strategies for protein depletion in this pathogen remain limited. Herein, we constructed a new CRISPR interference (CRISPRi) system for gene repression based on the combination of a shuttle vector pVv3 and the nuclease-null Cas9 variant (dead Cas9, or dCas9) from Streptococcus pyrogens. This CRISPRi is induced by adding both IPTG and arabinose. We showed that gene repression is scalable via the use of multiple sgRNAs. We also demonstrated that this gene repression can be precisely tuned by adjusting the amount of two different inducers and can be reversed by removing the inducers. This system provides a simple approach for selective gene repression on a genome-wide scale in V. parahaemolyticus. Application of this system will dramatically accelerate investigations of this bacterium, including studies of physiology, pathogenesis, and drug target discovery.