Staphylococcus aureus, a rapidly evolving gram-positive bacterium, poses a growing threat due to its arsenal of virulence determinants. Notably, S. aureus can acquire various antibiotic resistances, as seen in methicillin-resistant and vancomycin-resistant strains (MRSA and VRSA, respectively), rendering them resistant to conventional treatments. In response, CRISPR-Cas systems, which provide adaptive immunity to bacteria and archaea, have emerged as potential alternatives to conventional antibiotics. However, their implementation has been hindered by a lack of generalizable design rules for maximizing potency and minimizing resistance. To address this challenge, we evaluated an essential and defensive gene co-targeting approach as a design rule for developing effective CRISPR-Cas antimicrobials against S. aureus. This approach involves simultaneously targeting cellular processes necessary for survival and stress response. To achieve this, we first identified optimal guide RNAs for essential and defensive genes using a high-throughput library depletion assay. Next, we compared the lethality of top-performing guide RNAs in isolation and combination with each other. Finally, we determined the molecular drivers underlying CRISPR killing to inform the design of next-generation CRISPR-Cas treatments. This work provides valuable insights for designing potent CRISPR-based antimicrobials, advancing strategies to combat antibiotic-resistant S. aureus infections.
