Strain-resolved CRISPRi-Seq reveals conserved antibiotic vulnerabilities in Staphylococcus aureus

Staphylococcus aureus remains a major clinical threat due to rising antibiotic resistance and high rates of treatment failure. Deciphering the genetic responses to antibiotic pressure and identifying conserved vulnerabilities are essential steps toward developing broadly effective therapies. Here, we constructed strain-resolved CRISPR interference (CRISPRi) libraries targeting all genes in four clinically relevant S. aureus strains spanning major clonal complexes. CRISPRi-seq screens enabled high-resolution mapping of their fitness landscapes and the definition of a core essentialome representing robust targets for antimicrobial intervention. Exposure of the CRISPRi libraries to four mechanistically distinct antibiotics revealed genome-wide susceptibility profiles, identifying both strain-dependent and conserved susceptibility signatures shaped by the drug mode of action and genetic background. Analysis of these conserved vulnerabilities provided insight into antibiotic-specific stress responses and resistance mechanisms. Among the core determinants of vancomycin vulnerability, we identified several previously uncharacterized genes, including a conserved membrane-associated operon, here designated EsrABC, whose disruption markedly increases vancomycin sensitivity in the four strains. Our study provides a genome-wide atlas of S. aureus fitness and conditional vulnerabilities, fully explorable in the here-developed online AureoBrowse platform (https://aureobrowse.veeninglab.com/), revealing candidates for synergistic therapies and potential therapeutic targets.