Tandem crRNA array-mediated Cas12a platform for all-in-one multiplex detection of foodborne pathogens

Rapid multiplex detection of foodborne pathogens is critical for ensuring food safety and protecting public health, yet simultaneous identification of multiple species in samples remains challenging. Here, we present a tandem crRNA array-mediated Cas12a platform that enables compact and resource-efficient multiplex diagnostics. A dual crRNA array targeting Escherichia coli O157:H7 and Staphylococcus aureus was systematically optimized, functioning reliably at 300–500 nM Cas12a while reducing crRNA and reporter usage by 67% and 40%, respectively. Fluorescence output was enhanced by 30% following brief pre-assembly of Cas12a with the dual crRNA array, and stable performance was maintained at 35–39°C, supporting field adaptability. The dual array further revealed a positional effect optimized by a zero-nucleotide linker, enabling quantitative detection of both pathogens with excellent linearity ( R 2 > 0.99 at < 2 nM). Building on this principle, an octet array achieved one-pot detection of eight pathogens with balanced signals and high specificity. Collectively, these findings establish tandem crRNA arrays as a scalable and field-deployable framework, providing mechanistic insights into array design and enzyme loading strategies, while offering a cost-efficient and sustainable solution for high-throughput Cas12a diagnostics in food-safety monitoring. • Tandem crRNA arrays enable scalable and versatile multiplex Cas12a diagnostics. • Dual-array design improves detection efficiency while reducing reagent consumption. • Positional effects in arrays boost quantitative performance and inform design. • Rational Cas12a-to-crRNA ratio adjustment balances multiplex fluorescence signals. • Compact, cost-effective assay suitable for on-site food safety monitoring.