Genome-wide CRISPRi screens reveal the essentialome and determinants for susceptibility to dalbavancin in Staphylococcus aureus
Antibiotic resistance remains a major problem for treatment of staphylococcal infections. Knowing which genes influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. In this work, we develop a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrate its use by quantifying the essentialome in different strains through CRISPRi-seq, and use it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we found genes previously reported to be involved in antibiotic susceptibility, but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional knock-out approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods to the field. Notably, the non-essential pbp4 offers protection against dalbavancin, shedding light on the known synergism between this drug and PBP-targeting beta-lactam antibiotics. Knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, while knockdown of the Shikimate pathway surprisingly has the opposite effect. The results presented here demonstrate the potential of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility, grant insights into dalbavancin-specific mechanisms of action and resistance development, and pave the way to explore alternative antimicrobial treatments through these insights.