Ultrasensitive detection of methicillin-resistant Staphylococcus aureus using a T7 exonuclease-assisted PAM-free dual CRISPR-Cas12a biosensor

Methicillin-resistant Staphylococcus aureus (MRSA) strains are prevalent foodborne superbugs and pose significant pathogens for both humans and animals. The sensitive and specific diagnosis of MRSA holds paramount importance for its prevention and treatment. Herein, we develop a T7 exonuclease-assisted PAM-free Dual CRISPR-Cas12a Biosensor (TDCB) targeting the antibiotic resistance gene MecA for ultrasensitive and highly specific MRSA detection. After generation of single-stranded DNA (ssDNA) obtained with T7 exonuclease digestion of amplified MRSA genome, the trans-cleavage activity of dual-Cas12a-crRNA complexes was initiated without the requirement of protospacer adjacent motifs (PAM). The fluorescent signal obtained with TDCB biosensor was stronger, compared with the single-Cas12a biosensor. TDCB detects the MRSA genome and cells with a high sensitivity of 100 ag/mL and 1 cfu/mL, respectively. Moreover, TDCB can distinguish MRSA from tested 6 non-MRSA bacterial species from each other. We further validated TDCB using MRSA-infected tilapia fishes and obtained results consistent with the qPCR assay. This TDCB technology, a novel Cas12a-based biosensor that detects genomic regions without reliance on PAM motifs, can have very broad utilization in hospitals, food and feed industry, and fish and dairy farming.