Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 741 751 Arooj Khan Institute of Molecular Biology and Biotechnology, University of Lahore, Lahore, Pakistan Adnan Iqbal Institute-National Research Institute, Radzikow, Blonie, Poland Husnain Athar Department of Pediatrics, Punjab Institute of Cardiology, Lahore, Pakistan Ahsan Sheikh Institute of Molecular Biology and Biotechnology, University of Lahore, Lahore, Pakistan; Centre for Research in Molecular Medicine (CRiMM), University of Lahore, Lahore, Pakistan 2025 11 20 2026 07 08 Background and Objectives: The global rise of antimicrobial resistance threatens effective treatment of bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen whose resistance to β-lactam antibiotics is primarily mediated by the mecA gene, encoding the low-affinity penicillin-binding protein PBP2a. This study aimed to investigate whether CRISPR-dCas9-mediated targeting of mecA could suppress resistance-associated gene expression and restore β-lactam susceptibility in MRSA. Materials and Methods: Seven target sites within mecA, comprising two non-coding and five coding regions, were selected for CRISPR-dCas9 targeting. The effects of gene interference were evaluated using antibiotic disk diffusion assays, minimum inhibitory concentration (MIC) testing with gradient E-strips, RT-PCR, and Sanger sequencing. Results: Targeting non-coding regions suppressed mecA transcription and reduced PBP2a production, whereas targeting coding regions generated mutations associated with dysfunctional PBP2a. CRISPR-dCas9 treatment significantly reduced mecA expression and increased MRSA susceptibility to β-lactam antibiotics, particularly ampicillin and cefixime. MIC values decreased by up to 12-fold and 6-fold, respectively (p<0.05). Conclusion: CRISPR-dCas9-mediated targeting of mecA effectively reduced β-lactam resistance in MRSA. This approach demonstrates potential for precision-based antimicrobial resistance management and warrants further investigation as a strategy for combating drug-resistant bacterial infections. https://ijm.tums.ac.ir/index.php/ijm/article/view/5951 https://ijm.tums.ac.ir/index.php/ijm/article/download/5951/1935