A programmable, selection-free CRISPR interference system in Staphylococcus aureus for long-term host interaction...

Common dCas9-based CRISPR interference (CRISPRi) systems for manipulating bacterial gene expression require antibiotic selection and exogenous inducer molecules, limiting their applicability in infection models. For Staphylococcus aureus, we have developed a programmable, selection-free CRISPRi system leveraging the pCM29 plasmid, which is stable without antibiotic selection. In this system, dCas9 expression is regulated by an endogenous promoter, and sgRNA expression is driven by a constitutive promoter, eliminating the need for exogenous inducer molecules. We programmed the system to silence the expression of the coagulase or autolysin genes whenever their respective endogenous promoters were activated. We confirmed selection-free interference with target gene expression for ≥27 generations by qPCR and protein target-dependent in vitro or in vivo phenotypic assays (plasma coagulation, THP-1 cell, and Galleria mellonella infection). The system is suitable for interrogating gene function in long-term studies of S. aureus pathogenesis and represents a blueprint for similar CRISPRi systems in other species.