Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a critical public health threat due to its broad-spectrum antimicrobial resistance and capacity for horizontal gene transfer. Three clinical CRKP isolates, each carrying one of the three major classes of carbapenemase as class A ( bla KPC ), class B ( bla NDM ), and class D ( bla OXA ) were selected. A CRISPR/Cas9-based system (pCasKP-pSGKP) was employed to target carbapenem resistance genes in these strains (KP21040 with bla OXA-181 , KP4-78 with bla NDM-1 , and KP5-4 with bla KPC-2 ). CRISPR/Cas9-mediated editing led to partial reduction or complete loss of resistance plasmids, as evidenced by S1 nuclease-pulsed-field gel electrophoresis. This plasmid elimination correlated with a marked restoration of susceptibility to ertapenem, showing a greater than 64-fold reduction in minimum inhibitory concentrations (MICs) across all strains. In KP21040, MICs for ertapenem and levofloxacin decreased to 0.006 μg/mL and 0.125 μg/mL, respectively. Whole-genome analysis revealed that bla OXA-181 was flanked by insertion sequence (IS) 26 elements, which mediated homologous recombination upon CRISPR-induced double-strand breaks, resulting in excision of a ∼15 kb segment including bla OXA-181 and qnrS1 . These findings suggest that ISs may enhance CRISPR efficacy by promoting recombination-driven deletion. Moreover, the complete removal of all three resistance plasmids was observed in the KP5-4 strain harboring bla KPC-2 . This study demonstrates that CRISPR/Cas9-based genome editing can eliminate plasmid-encoded carbapenemase genes in clinical CRKP isolates and, in specific genetic contexts, facilitate the concurrent removal of associated quinolone resistance determinants. These findings support CRISPR-based genome editing as a proof-of-concept strategy for addressing plasmid-mediated multidrug resistance in Gram-negative pathogens. • A clustered regularly interspaced palindromic repeats was used to modify carbapenem resistance genes in clinical carbapenem-resistant Klebsiella pneumoniae isolates. It effectively restored carbapenem susceptibility and may provide a framework to assess plasmid-associated gene functions in clinical isolates.
Kuo et al. (Sun,) studied this question.