Elimination of blaKPC−2-mediated carbapenem resistance in Escherichia coli by CRISPR-Cas9 system

The purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the bla.sub.KPC-2 gene by curing the bla.sub.KPC-2-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system. The single guide RNA (sgRNA) specifically targeted to the bla.sub.KPC-2 gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(bla.sub.KPC-2) was transformed into Escherichia coli (E.coli) carrying pET24-bla.sub.KPC-2. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. In the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the bla.sub.KPC-2 gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear bla.sub.KPC-2-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the bla.sub.KPC-2-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. In conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the bla.sub.KPC-2 gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.