Genome-wide CRISPRi-seq maps essential-gene contributions to aminoglycoside susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen whose multiple intrinsic and acquired antibiotic resistance mechanisms restrict treatment options. Essential genes offer potential antimicrobial targets, but their vulnerability to partial inhibition and contribution to antibiotic susceptibility remain incompletely characterized. Here, we developed a rhamnose-inducible CRISPR interference (CRISPRi) platform and an 83,078-guide library for genome-wide screening in PAO1, with mismatch guides enabling graded repression. Fitness profiling in antibiotic-free Mueller–Hinton medium identified 743 genes whose repression reduced competitive fitness and revealed sequence features associated with guide performance. Combining full-match and mismatch-guide responses revealed substantial variation in essential-gene fitness effects across predicted repression strengths, while comparison of fitness profiles between Mueller–Hinton medium and fetal bovine serum identified condition-dependent effects alongside a conserved fitness landscape. Under aminoglycoside exposure, we identified 118 recurrent sensitizing and 120 recurrent protective gene knockdowns, including many targeting essential and fitness-associated functions. Growth and killing assays with individual strains confirmed that CRISPRi targeting of atpI , accC , secA and ftsZ enhanced tobramycin activity. Finally, transcriptome analysis of these knockdowns confirmed target silencing, captured polar effects on neighboring genes and revealed distinct but overlapping transcriptional responses. Together, these findings quantify the variable fitness costs of essential-gene repression and identify candidate targets for potentiating aminoglycoside activity.