Single-Tube Dual-Gene Detection of Methicillin-Resistant Staphylococcus aureus via Selective Trans -Cleavage Preferences of Cas9 and Cas12a

Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing infections. Current dual-gene detection methods based on CRISPR-Cas systems often require additional transcription steps, which increase the complexity of the assay and extend the turnaround time. Here, we report a single-tube dual-gene detection platform that leverages the orthogonal trans-cleavage preferences of Cas9 and Cas12a. By exploiting the loss of RNA cleavage activity in Cas12a when guided by split crRNA, and the inability of Cas9 to cleave DNA hairpin probes, we established a single-tube assay capable of simultaneously detecting the MRSA resistance gene (mecA) and the S. aureus-specific nuclease gene (nuc). The platform achieved attogram-level sensitivity and single-cell detection with high specificity against non-MRSA strains. Validation in an in vivo tilapia infection model demonstrated complete concordance with qPCR, reaching 100% positive and negative percent agreements. This work presents a streamlined, accurate, and practical approach for dual-gene pathogen detection, expanding the potential of Cas protein orthogonality for multiplex diagnostics.