Dual-lock gated DNAzyme-CRISPR cascade for amplification-free ultrasensitive profiling of FTO demethylase activity.

N6-methyladenosine, the most prevalent internal mRNA modification in eukaryotes, dynamically regulates transcriptomic homeostasis via FTO-mediated demethylation. CRISPR-based FTO detection technologies represent a significant research direction. However, most current strategies depend critically on nucleic acid pre-amplification steps, which introduce substantial risks of amplification leakage, thereby compromising their ability to achieve stable, high-sensitivity FTO detection. To address this, we designed a dual-lock gated DNAzyme-CRISPR cascading platform that integrates a double-locked DNAzyme with a redundant structure regulated CRISPR-Cas12a trans-cleavage signal amplification system. This architecture achieves an unprecedented limit of detection of 0.083 pM, which is 12 times more sensitive than commercial ELISA kits. Clinical validation using 20 breast cancer patient tissues demonstrated robust cancer/normal discrimination (AUC = 0.9853) and significant FTO upregulation in triple-negative subtypes (p < 0.001). The modular design establishes a universal framework for epigenetic enzyme detection while enabling dynamic monitoring of tumor epigenetic remodeling.