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 (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 , , and in various species of , , , , and clinical isolates, with a >94% curing efficiency. In addition, we also demonstrated that pCasCure can efficiently eliminate several epidemic carbapenem-resistant plasmids, including the -harboring IncFIIK-pKpQIL and IncN pKp58_N plasmids, the -harboring pOXA-48-like plasmid, and the -harboring IncX3 plasmid, by targeting their replication and partitioning ( in pKpQIL) genes. However, curing the gene failed to eliminate its corresponding pOXA-48-like plasmid in clinical isolate 49210, while further next-generation sequencing revealed that it was due to IS -mediated recombination outside the CRISPR-Cas9 cleavage site resulting in truncation and, therefore, escaped plasmid curing. Nevertheless, the curing of carbapenemase genes or plasmids, including the truncation of 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.