The genetic landscape of antibiotic sensitivity in Staphylococcus aureus

ABSTRACT A comprehensive genetic landscape of antibiotic sensitivity in Staphylococcus aureus is lacking. Using ultra-dense CRISPR-interference libraries, we systematically quantified global gene fitness across ten antibiotics and uncovered hundreds of significant antibiotic-gene interactions. Essential genes dominated these interactions, a finding not revealed by transposon-based studies. Processes most vulnerable to transcriptional repression under antibiotic conditions included cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), coenzyme A biosynthesis, and riboflavin metabolism. Network and genetic analyses further revealed novel synergistic genetic interactions (GIs) within these processes, including an extensive CC-DD subnetwork. Only a subset of CC-DD synergies was dependent on the cell division inhibitor SosA. Informed by these GIs, we identified multiple drug-drug combinations with potent synergistic activity against multidrug-resistant S. aureus . Our detailed profiling of drug-gene, gene-gene, and drug-drug interactions reveals novel functional relationships among essential genes and defines a vulnerability landscape to guide new drug target discovery and effective combination therapies.