Functional Analyses of Cassette Chromosome Recombinase C2 (CcrC2) and Its Use in Eliminating Methicillin Resistance by Combining CRISPR–Cas9

Worldwide occurrence of methicillin-resistant Staphylococcus aureus (MRSA) poses enormous challenges for both communities and health care settings. Cassette chromosome recombinases (Ccr) specifically perform excision and acquisition of a staphylococcal cassette chromosome mec (SCCmec) in staphylococci and are responsible for the spread of methicillin resistance. This study explored the roles of CcrC2, a recently discovered Ccr, in the horizontal transfer of SCCmec and developed a potential means to control the spread of methicillin resistance. Knockout of CcrC2 completely aborted the excision of SCCmec, while overexpression of CcrC2 partially removed the SCCmec from the genome and transformed methicillin-resistant Staphylococcus aureus (MRSA) into methicillin-susceptible Staphylococcus aureus (MSSA). Moreover, two nucleotide residues (G5C6) in the direct repeat sequence within an att site were found to be critical for excision and acquisition efficiencies. To block the horizontal transfer of methicillin resistance, a SCCmec killer system was developed by combining the CcrC2-mediated SCCmec excision and the mecA-targeting CRISPR–Cas9 machinery. The SCCmec killer transformed MRSA to MSSA and disrupted the mecA-carrying SCCmec intermediate, thereby eliminating methicillin resistance determinant mecA gene inside a MRSA cell and blocking the horizontal transfer of SCCmec. The SCCmec killer was versatile for efficiently removing multiple types of SCCmec elements. It is envisioned that this approach could offer a new means to control the spread of methicillin resistance