Cleavage-responsive DNA/AgNCs enable accelerated Cas12a trans-cleavage for rapid multigene methylation diagnosis

DNA methylation, particularly 5-methylcytosine (5 mC), is a key epigenetic modification involved in the regulation of gene expression and genomic stability, and has emerged as a promising biomarker for early cancer screening and molecular stratification. CRISPR-Cas12a systems have been increasingly exploited to convert methylation-associated sequence information into detectable signals owing to their programmability and collateral cleavage-mediated signal amplification. However, many CRISPR-based assays remain constrained by the high background and relatively slow response kinetics of conventional fluorophore-quencher reporters. To overcome these limitations, we developed a cleavage-responsive DNA-templated silver nanocluster (DNA/AgNC) reporter that translates Cas12a trans-cleavage activity into a green-to-red ratiometric fluorescence shift. In this design, the AgNC-templating DNA scaffold itself serves as an enzymatically cleavable signal transducer, rather than relying on a terminal fluorophore-quencher pair. Compared with a representative F-ssDNA-Q reporter, the DNA/AgNC reporter exhibited stronger apparent association with Cas12a and an approximately two-fold improvement in apparent catalytic efficiency. When incorporated into an MSRE-RPA-Cas12a workflow, the platform achieved a limit of detection of 74.5 aM, while completing the Cas12a reporting step within 30 min. Coupling this assay with a miniaturized optoelectronic device further enabled spatially resolved profiling of five genomic loci, with relative errors of approximately 5%. Overall, this strategy establishes a ratiometric reporter format for CRISPR-based DNA methylation profiling and offers potential for point-of-care epigenetic biosensing.