A programmable, selection-free CRISPR interference system in Staphylococcus aureus

Common dCas9-based CRISPR interference (CRISPRi) system for gene regulation requires antibiotic selection and exogenous inducer molecules, posing significant challenges when applied in in vivo bacterial infection models. Using Staphylococcus aureus as a model organism, we have developed a programmable, plasmid-based, but selection-free (ppsf)-CRISPRi system that is based on the pCM29- plasmid which is stable without antibiotic selection. In this ppsf-CRISPRi system, dCas9 expression is regulated by an endogenous virulence gene promoter, and sgRNA expression is driven by a constitutive promoter eliminating the need for exogenous inducer molecules. The system was programmed to silence the expression of genes encoding the virulence factor coagulase or peptidoglycan hydrolase autolysin, whenever their respective endogenous promoter was activated. The selection-free functionality was confirmed over at least 27 generations and verified by qPCR and phenotypic assays depending on the protein target, including coagulation of rabbit plasma and THP-1 macrophage cell infection in vitro as well as in vivo infection of Galleria mellonella larvae, in each case phenocopying the observations made using transposon mutant strains. The system is suitable for long-term studies of S. aureus pathogenesis in vitro or in vivo and represents a blueprint for the development of similar ppsf-CRISPRi systems in other bacterial species.