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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.