Allosteric Aptamer CRISPR/Cas Activation Enables Non-competitive ATP Detection and Meat Freshness Assessment

CRISPR/Cas-based aptasensors (Cas-aptasensors) hold great promise for detecting non-nucleic acid targets, yet their intrinsic competitive recognition mechanism imposes a trade-off between transduction efficiency and background leakage. In this study, we developed a Cas-aptasensor that employs a non-competitive recognition mechanism. In our design, the aptamer-target interaction accelerates the toehold-mediated strand displacement reaction and exposes a second toehold domain. The CRISPR/Cas system is ultimately activated via a cascade strand-displacement reaction, which is hindered in the absence of the target and, thus, delays activation. We demonstrated the applicability of this non-competitive Cas-aptasensor for the detection of ATP, achieving a detection limit as low as 1.0 nM within 45 min. Furthermore, we successfully applied this method to ATP detection in complex matrices and to assess the freshness of diverse meat products across different storage temperatures. Overall, this work advances the design of Cas-aptasensors and expands their potential applications in food safety monitoring.