CRISPR Interference-Based Gene Silencing of Virulence Regulators in Multidrug-Resistant Staphylococcus aureus: A Novel Anti-Virulence Strategy for Nigerian Clinical Isolates

Multidrug-resistant Staphylococcus aureus infections are a major cause of morbidity and mortality in Nigerian hospitals, and the failure of conventional antimicrobial strategies to combat biofilm-embedded and methicillin-resistant strains necessitates exploration of anti-virulence approaches targeting regulatory networks rather than growth. This research exploits CRISPR interference technology to systematically silence virulence regulators in multidrug-resistant S. aureus clinical isolates from tertiary hospitals in Lagos and Ogun States, South West Nigeria, providing proof-of-concept for a novel anti-virulence therapeutic strategy. MDR and MRSA isolates with confirmed virulence gene carriage will be collected from clinical specimens over twelve months. CRISPRi constructs targeting key global virulence regulators including agr, sarA, saeRS, and codY will be designed and delivered by electroporation-optimised transformation protocols adapted for clinical strain backgrounds. Transcriptomic confirmation of target gene silencing by RNA-seq will verify CRISPRi efficiency. In vitro phenotypic assays will quantify the impact of regulator silencing on biofilm formation, haemolysin production, fibronectin binding, protease secretion, and intracellular invasion. In vivo efficacy will be assessed in Galleria mellonella infection models. Combinatorial CRISPRi silencing with sub-inhibitory antibiotic exposure will be evaluated for synergistic virulence attenuation. Original contributions include the first CRISPRi anti-virulence study on Nigerian clinical MRSA isolates, a novel framework for regulator-targeted therapeutic design applicable to Gram-positive nosocomial pathogens, and an empirical anti-virulence database for MDR S. aureus from West Africa. Keywords: CRISPRi, Staphylococcus aureus, anti-virulence, virulence regulators, MRSA