CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant <i>Staphylococcus aureus</i>

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), targeting methicillin (<i>mec</i>A), gentamicin (<i>aac</i>A), and ciprofloxacin (<i>grl</i>A, <i>grl</i>B) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in <i>mec</i>A, a 5.5-fold decrease in <i>grl</i>A, a 6-fold decrease in <i>grl</i>B, and a 4-fold decrease in <i>aac</i>A expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the <i>grl</i>B and <i>aac</i>A genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.