CRISPR-driven strategies to disrupt methicillin-resistant Staphylococcus aureus biofilms: a review

Methicillin-resistant Staphylococcus aureus exhibits heightened tolerance to antimicrobial therapy through restricted drug penetration, extracellular polymeric substance-mediated protection, metabolic heterogeneity, and persister cell formation, limiting the effectiveness of current treatment strategies. CRISPR-Cas systems have emerged as programmable antimicrobial tools capable of targeting resistance genes, virulence determinants, and regulatory pathways; however, existing approaches remain largely gene-centric and insufficiently integrated with the biological complexity of biofilm-associated infections. This review aims to provide a comprehensive and integrative analysis of CRISPR-based strategies for targeting MRSA biofilms by linking molecular CRISPR mechanisms with key biofilm processes and evaluating their translational potential. CRISPR-Cas systems have emerged as programmable antimicrobial platforms with the ability to selectively target resistance genes, virulence factors, and regulatory networks. In MRSA biofilms, these systems are increasingly being explored for their potential to disrupt biofilm-associated determinants and weaken the molecular basis of persistence. Recent advances involving Cas9, Cas12a, and Cas13 highlight the potential of CRISPR-based targeting to interfere with resistance mechanisms, quorum sensing pathways, and structural components relevant to biofilm stability. Emerging in vivo studies, particularly those using engineered bacteriophages and localized delivery systems, provide early evidence that CRISPR-based strategies can reduce bacterial burden and impair biofilm integrity under physiologically relevant conditions. Nevertheless, significant barriers remain, including limited penetration into mature biofilms, delivery inefficiency, off-target activity, immunogenicity, resistance evolution, and regulatory uncertainty. Ultimately, CRISPR-based interventions represent a promising but still developing approach for the control of MRSA biofilm-associated infections, requiring further refinement in delivery design, target selection, and translational validation.