BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a critical public health threat due to its broad-spectrum antimicrobial resistance and capacity for horizontal gene transfer.
METHODS: Three clinical CRKP isolates, each carrying one of the three major classes of carbapenemase as class A (bla), class B (bla), and class D (bla) were selected. A CRISPR/Cas9-based system (pCasKP-pSGKP) was employed to target carbapenem resistance genes in these strains (KP21040 with bla, KP4-78 with bla, and KP5-4 with bla). KPC NDM OXA OXA-181NDM-1KPC-2
RESULTS: 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 blawas 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 blaand 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. OXA-181OXA-181KPC-2
CONCLUSION: 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.
CRISPR Gene Editing