Lipoplex as a useful tool for resensitization of methicillin-resistant Staphylococcus aureus to erythromycin via CRISPR-Cas technology.

The global escalation of antimicrobial resistance (AMR) necessitates innovative therapeutic interventions. Methicillin-resistant Staphylococcus aureus (MRSA) remains a primary clinical threat due to its extensive resistance profile. In this study, CRISPR-Cas9 technology was employed to target the ermA gene, which is responsible for erythromycin resistance in clinical MRSA isolates. A cationic liposome (Lip) composed of cholesterol, DOTAP, and DOPE was developed to deliver the pCasSA plasmid. Particle characterization revealed stable liposomes (Lips) (<200 nm, PDI < 0.3, ZP>+30 mV). pCasSA was loaded into the Lip, to form a lipoplex (Lpx), a complex of Lip and pCasSA, via electrostatic interactions. Cytotoxicity studies using mouse fibroblast L929 cells revealed that cell viabilities remained at approximately 93% and 83% following treatment with the highest concentration of Lip and Lpx formulations, respectively. Successful gene editing was confirmed via Sanger sequencing and RT-qPCR, showing a 75% reduction in ermA expression when Lpx wascombined with sonoporation. Phenotypic assays demonstrated a significant restoration of susceptibility. The minimum inhibitory concentration (MIC) of erythromycin decreased 8-fold (from 8 mg/L to 1 mg/L), and inhibition zones increased 2.5-fold (from 12 mm to 30 mm). These findings suggest that Lpx-mediated CRISPR-Cas9 delivery is a highly efficient strategy for antibiotic resensitization, potentially restoring the clinical utility of existing antimicrobials.