Precision Genome Editing Unveils a Breakthrough in Reversing Antibiotic Resistance: CRISPR/Cas9 Targeting of Multi-Drug Resistance Genes in Methicillin-ResistantStaphylococcus aureus

Abstract

AbstractAntibiotic resistance poses a global health crisis, limiting the efficacy of available therapeutic agents and hindering the development of new antibiotics. The pharmaceutical industry’s waning interest in antibiotic production further exacerbates this challenge. Addressing antibiotic resistance demands innovative solutions. Here, we explore the application of CRISPR-Cas-based antimicrobials as a pioneering approach to combat multidrug resistance. Our study focuses on the methicillin-resistant clinical Staphylococcus aureus (MRSA), a significant clinical threat. Utilizing CRISPR/Cas9 technology, we aimed to concurrently target methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes, thereby altering the resistance profile and enhancing sensitivity to antibiotics. We engineered CRISPR plasmids containing sgRNAs specific to the target regions, which were subsequently electroporated into MRSA strains. Real-time Polymerase Chain Reaction (RT-PCR) assessed changes in resistance gene expression, while disk diffusion and broth microdilution methods determined alterations in resistance status. Western blotting analyzed changes in PBP2a expression, and Sanger sequence analysis confirmed mutations in target regions. Results revealed a statistically significant 1.5-fold decrease in mecA gene expression, 5.5-fold decrease in grlA gene, 6-fold decrease in grlB gene, and 4-fold decrease in aacA gene compared to the wild type strain, as determined by RT-PCR. Antibiotic susceptibility tests demonstrated the suppression of mecA, grlA, grlB, and aacA genes, resulting in the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis showed 70% decrease in PBP2a expression, indicating the breakdown of beta-lactam resistance. Sanger sequence analysis confirmed point mutations in grlB and aacA genes, along with three-base mutations in grlA and mecA genes. Our findings underscore the potential of CRISPR/Cas9 technology to mitigate antibiotic resistance, offering a transformative strategy to restore the efficacy of existing antibiotics in the face of multidrug-resistant pathogens.