CRISPR-dCas9-mediated knockdown ofprtR, an essential gene inPseudomonas aeruginosa

Pseudomonas aeruginosais 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 inP. aeruginosa. To establish a gene interference platform inP. 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 geneprtRthat is essential for the viability ofP. aeruginosa. Cells expressing theprtR-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-mediatedprtRinhibition indicated thatprtRis a global regulator affecting multiple biological processes. In conclusion, the CRISPR-dCas9-based gene knockdown system has been successfully implemented inP. aeruginosaand demonstrated to be an effective tool in the investigation of essential or difficult-to-inactivate genes in this species.