CRISPR-Cas9-mediated carbapenemase gene and plasmid curing in carbapenem-resistant enterobacteriaceae

Combating plasmid-mediated carbapenem resistance is essential to control and prevent the dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Here, we conducted a proof-of-concept study to demonstrate that CRISPR-Cas9-mediated resistance gene and plasmid curing can effectively resensitize CRE to carbapenems. A novel CRISPR-Cas9-mediated plasmid-curing system (pCasCure) was developed and electrotransferred into various clinical CRE isolates. The results showed that pCasCure can effectively cure blaKPC, blaNDM, and blaOXA-48 in various Enterobacteriaceae species of Klebsiella pneumoniae, Escherichia coli, Enterobacter hormaechei, Enterobacter xiangfangensis, and Serratia marcescens clinical isolates, with a >94% curing efficiency. In addition, we also demonstrated that pCasCure can efficiently eliminate several epidemic carbapenemresistant plasmids, including the blaKPC-harboring IncFIIK-pKpQIL and IncN pKp58_N plasmids, the blaOXA-48-harboring pOXA-48-like plasmid, and the blaNDM-harboring IncX3 plasmid, by targeting their replication and partitioning (parA in pKpQIL) genes. However, curing the blaOXA-48 gene failed to eliminate its corresponding pOXA-48-like plasmid in clinical K. pneumoniae isolate 49210, while further next-generation sequencing revealed that it was due to IS1R-mediated recombination outside the CRISPR-Cas9 cleavage site resulting in blaOXA-48 truncation and, therefore, escaped plasmid curing. Nevertheless, the curing of carbapenemase genes or plasmids, including the truncation of blaOXA-48 in 49210, successfully restore their susceptibility to carbapenems, with a >8-fold reduction of MIC values in all tested isolates. Taken together, our study confirmed the concept of using CRISPR-Cas9-mediated carbapenemase gene and plasmid curing to resensitize CRE to carbapenems. Further work is needed to integrate pCasCure in an optimal delivery system to make it applicable for clinical intervention.