Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa

Abstract

Summary: Despite being fundamentally important and having direct therapeutic implications, the functional genomics of the clinical isolates of multidrug-resistant (MDR) pathogens is often impeded by the lack of genome-editing tools. Here, we report the establishment of a highly efficient, in situ genome-editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems. Using this approach, we generate various reverse mutations in an epidemic MDR genotype, PA154197, and identify underlying resistance mechanisms that involve the extensive synergy among three different resistance determinants. Screening a series of “ancestor” mutant lines uncovers the remarkable sensitivity of the MDR line PA154197 to a class of small, cationic peptidomimetics, which sensitize PA154197 cells to antibiotics by perturbing outer-membrane permeability. These studies provide a framework for molecular genetics and anti-resistance drug discovery for clinically isolated MDR pathogens. : Xu et al. develop an efficient genome-editing technique in genetically recalcitrant clinical and environmental P. aeruginosa strains by exploiting their endogenous type I-F CRISPR-Cas system. Employing the technique, they identify underlying resistance mechanisms of an epidemic MDR genotype, PA154197, efficiently and develop anti-resistance strategies based on collateral sensitivity. Keywords: multidrug resistance, Pseudomonas aeruginosa, native CRISPR-Cas system, collateral sensitivity, cationic peptidomimetics, genome editing, multidrug efflux pump, anti-resistance drug discovery