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

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 , 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 or phenotypic assays (plasma coagulation, THP-1 cell, and infection). The system is suitable for interrogating gene function in long-term studies of pathogenesis and represents a blueprint for similar CRISPRi systems in other species.