Biomedical journal · 2026
CRISPR-Cas9-mediated elimination of plasmid-borne carbapenemase genes restores ertapenem susceptibility in clinical Klebsiella pneumoniae isolates.
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Kuo Shu-Fang, Huang Tsung-Yu, Lee Chih-Yi, Chen Fang-Ju, Lee Chen-Hsiang
Аннотация
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 (blaKPC), class B (blaNDM), and class D (blaOXA) were selected. A CRISPR/Cas9-based system (pCasKP-pSGKP) was employed to target carbapenem resistance genes in these strains (KP21040 with blaOXA-181, KP4-78 with blaNDM-1, and KP5-4 with blaKPC-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 blaOXA-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 blaOXA-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 blaKPC-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.
METHODS: Three clinical CRKP isolates, each carrying one of the three major classes of carbapenemase as class A (blaKPC), class B (blaNDM), and class D (blaOXA) were selected. A CRISPR/Cas9-based system (pCasKP-pSGKP) was employed to target carbapenem resistance genes in these strains (KP21040 with blaOXA-181, KP4-78 with blaNDM-1, and KP5-4 with blaKPC-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 blaOXA-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 blaOXA-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 blaKPC-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.