Li Wan, Menghan Liu, Panos Oikonomou, Sydney Blattman, Mirela Berisa, Falguni Paul, Julia Hettleman, Joseph Gonzalez, Qiaoyu Hu, Howe Chen, Saeed Tavazoie, Wenyan Jiang
A comprehensive genetic landscape of antibiotic sensitivity in Staphylococcus aureus is lacking. Using genome-scale CRISPR-interference libraries, we systematically quantified global gene fitness across 10 antibiotics and uncovered hundreds of significant antibiotic-gene interactions. Essential genes dominated these interactions, a finding not revealed by transposon-based studies. CRISPR interference repression of transcriptional and translational processes desensitized bacteria to multiple antibiotics. In contrast, repression of cell wall synthesis/cell division (CC), DNA replication/DNA recombination (DD), coenzyme A biosynthesis, and riboflavin metabolism strongly sensitized bacteria to antibiotics. Network and genetic analyses further revealed synergistic genetic interactions (GIs) within these bioprocesses, 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 important functional relationships among essential genes and defines a vulnerability landscape to guide drug target discovery and effective combination therapies.