Pseudomonas aeruginosa (Pae) is a major opportunistic human pathogen, classified by the WHO in 2024 as a high-priority antibiotic resistant threat. It causes severe infections in immunocompromised individuals and patients with cystic fibrosis, largely due to a combination of intrinsic and acquired resistance mechanisms. CRISPR interference (CRISPRi) enables programmable, transcriptional repression of target genes and is well-suited for genome-wide interrogation of gene-drug interactions, allowing identification of essential pathways that become particularly vulnerable under antibiotic pressure. In CRISPRi, a single guide RNA (sgRNA) directs a catalytically inactive Cas9 protein (dCas9) to a target sequence adjacent to a PAM site, blocking RNA polymerase binding or elongation.

Here, we performed a genome-wide CRISPRi screen in the P. aeruginosa PAO1 reference strain, using an sgRNA pooled library exposed to sub-inhibitory concentrations of 12 antibiotics commonly used against Pae: aminoglycosides, β-lactams (including carbapenems), fluoroquinolones, and polymyxins. The analysis identified essential gene-drug interactions and pinpointed pathways (e.g., cell wall synthesis, cell division, fatty acids and lipopolysaccharide biosynthesis) whose repression altered antibiotic susceptibility. These genes represent potential therapeutic targets or adjuvant candidates to enhance existing antimicrobial efficacy while reducing toxicity associated with high drug doses. Overall, this platform provides a robust approach for investigating essential genes and their role in resistance, offering a promising strategy to identify pathogen vulnerabilities.

This work is supported by the Polish National Science Centre grant 2021/43/D/NZ2/02151.