CRISPR-dCas9-mediated knockdown of prtR, an essential gene in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a widely distributed non-fermentative Gram-negative opportunistic pathogen that is often responsible for nosocomial infections. Gene interference is a potentially valuable tool for investigating essential genes in P. aeruginosa. To establish a gene interference platform in P. aeruginosa, CRISPR system was used with an inactive Cas9 protein. The CRISPR-dCas9 system was cloned into pHERD20T, a shuttle vector with arabinose inducible promoter, and was further modified to target a regulatory gene prtR that is essential for the viability of P. aeruginosa. Cells expressing the prtR-targeting CRISPR interference (CRISPRi) showed growth defect in an arabinose dose-dependent manner. A high-throughput RNA sequencing analysis of bacterial cells with or without the CRISPRi-mediated prtR inhibition indicated that prtRis a global regulator affecting multiple biological processes. In conclusion, the CRISPR-dCas9-based gene knockdown system has been successfully implemented in P. aeruginosa and demonstrated to be an effective tool in the investigation of essential or difficult-to-inactivate genes in this species.