Intrinsic crRNA Scaffold Dynamic Reassembly Powered Cas12a Cascade Amplification for Multiplexed Detection

The reassembly of the intrinsic crRNA scaffold with complementary ssDNA activates Cas12a trans-cleavage activity, thereby overcoming the limitations of conventional split crRNA engineering strategies and resulting in expanded target detection range, reduced background interference, and improved signal-to-noise ratio. Here, we have developed a novel scaffold RNA reassembly biosensor that ingeniously couples catalytic nucleic acid (ribozyme or DNAzyme) cleavage as a signal transduction element with the Cas12a cascade amplification, enabling highly sensitive detection of diverse targets including miRNAs, small molecules, and metal ions. The system employs an intrinsic amplifier that integrates signal output with crRNA regeneration. Each Cas12a cleavage event not only generates a fluorescent signal but also releases scaffold RNA, which promptly reassembles into functional crRNA complexes, thereby driving continuous Cas12a activation. This crRNA scaffold dynamic reassembly circuit enables cascade signal amplification without requiring additional crRNA or activators, thereby facilitating one-pot detection with shortened reaction time, while simultaneously enhancing detection sensitivity and minimizing aerosol contamination risks. Experimental results demonstrated excellent sensitivity, specificity, and robustness in complex matrices such as serum and environmental water samples. Moreover, the system achieved accurate miRNA detection in different cell lines and clinical samples, which was corroborated by lateral flow analysis and showed strong agreement with the RT-qPCR gold-standard method. Owing to its modular design and powerful amplification capacity, this platform holds broad potential for versatile biosensing applications.