FIG 1.

Plasmid map of pEMPTY::sgRNA2 and the plasmid curing process in S. aureus using the pEMPTY::sgRNA2 system. (A) The shuttle vector pEMPTY::sgRNA2 contains the cas9 gene downstream of the tetracycline-inducible promoter P_xyl/tetO to guarantee regulation of curing functionality, the temperature-sensitive origin of replication pE194_ts-ori, which allows the loss of the vector system by increasing the temperature to 37°C, and a small guide RNA, consisting of guide RNA 2, which targets S. aureus plasmids fused to the scaffold tracrRNA sequence, downstream of the constitutive SP01 promoter. (B) pEMPTY::sgRNA2 is transformed into a S. aureus strain containing the target plasmid for curing, and transformants are selected on medium containing chloramphenicol as a selective agent. Cells are transferred into liquid TSB medium containing chloramphenicol and anhydrotetracycline (ATc), and the culture is incubated overnight at 28°C to allow cas9 gene expression and, thus, cleavage of target plasmids directed by guide RNA 2. The culture is plated on TSA containing chloramphenicol and antibiotic (cadmium, lincomycin, or erythromycin) selective TSA, and cured cells are identified by antibiotic sensitivity and PCR screening. A colony of cured cells is incubated overnight in nonselective TSB medium at 37°C, and the culture is plated on nonselective TSA and incubated overnight at 37°C to select a cured colony that has lost plasmid pEMPTY::sgRNA2.