CRISPR-Cas systems as next-generation antimicrobials: a systemic review of mechanisms, delivery strategies, and translational challenges

Introduction: The rapid global increase in multidrug-resistant (MDR) bacteria has compromised the effectiveness of conventional antibiotics, stressing the urgent need for alternative antimicrobial strategies. CRISPR-Cas systems, originally evolved as bacterial adaptive immune mechanisms, provide programmable and highly specific tools for targeting antimicrobial resistance (AMR) determinants. Objective: This systematic review aims to evaluate the antibacterial mechanisms, delivery strategies, preclinical evidence, safety considerations, and translational potential of CRISPR-Cas systems for combating MDR bacterial infections. Methods: experimental or preclinical investigations assessing CRISPR-Cas systems (Cas9, Cas12, Cas13, or related effectors) for antibacterial activity or antibiotic resensitization. Data were extracted on CRISPR effector type, bacterial target, delivery platform, and therapeutic outcome. Due to methodological heterogeneity, results were synthesized narratively. Results: Most studies reported effective killing or resensitization of MDR bacteria through chromosomal double-strand break induction, resistance plasmid curing, integron disruption, or RNA-targeted cleavage. Cas9 was the most frequently employed effector, followed by Cas12 and Cas13. Delivery strategies included bacteriophages, conjugative plasmids, and nanoparticle-based systems, with phage-mediated delivery demonstrating the most consistent efficacy in complex environments and animal models. Notably, a CRISPR-enhanced engineered bacteriophage cocktail (LBP-EC01) has advanced to clinical evaluation. Discussion: Overall, the evidence supports CRISPR-Cas antimicrobials as a promising precision-based approach for addressing AMR. However, major barriers remain, including limited host range, instability in physiological environments, emergence of escape mutations, and insufficient data on off-target effects and long-term safety. Addressing these challenges through optimized delivery platforms, multiplex targeting strategies, and standardized safety and regulatory frameworks will be essential for clinical translation. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/view/CRD420261319789, identifier CRD4201319789.