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.

Common dCas9-based CRISPR interference (CRISPRi) system for gene regulation requires antibiotic sele