Direct microRNA detection via topologically engineered CRISPR/Cas12a cascade amplification assay

The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its high specificity, sensitivity and programmability. However, direct RNA detection is hindered by its inherent DNA-targeting trans-cleavage activity, which typically necessitates a reverse transcription amplification to convert RNA into DNA. Herein, we report a one-pot assay that enables direct detection of microRNA-21 without target amplification. The method employs a molecular switch probe (MSP) that recognizes miRNA-21 and activates Cas12a activity, along with an amplifier probe (AMP) that establishes a self-driven cascade signal amplification. This biosensor achieves a detection limit of 2.88 pM, with high accuracy (recovery of 95.5%-108.6%) and good precision (RSD: 2.35%-7.18%), and exhibits excellent specificity against homologous miRNAs. Using this assay, we successfully quantified the elevated levels of miRNA-21 in lung squamous carcinoma (H520) cells compared to normal bronchial epithelial cells (BEAS-2B). Furthermore, a methodological comparison with RT-qPCR revealed a similar trend between the two methods. This study provides a simple and reliable strategy for direct RNA detection using CRISPR/Cas12a.