Genome-wide CRISPRi screens reveal the essentialome and determinants for susceptibility to dalbavancin in Staphylococcus aureus

Abstract

Antibiotic resistance and tolerance remain a major problem for treatment of staphylococcal infections. Knowing genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrated its use by quantifying the essentialome in different strains through CRISPRi-seq, and used 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. Notably, 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 and pave the way to explore alternative antimicrobial treatments through these insights.