Amplification-free miRNA detection with CRISPR/Cas12a system based on fragment complementary activation strategy

CRISPR/Cas12a systems have been repurposed as powerful tools for developing next-generation molecular diagnostics due to their trans-cleavage ability. However, it was long considered that the CRISPR/Cas12a system could only recognize DNA targets. Herein, we systematically investigated the intrinsic trans-cleavage activity of the CRISPR/Cas12a system (LbCas12a) and found that it could be activated through fragmented ssDNA activators. Remarkably, we discovered that the single-stranded DNA (ssDNA) activators in the complementary crRNA-distal domain could be replaced by target miRNA sequences without the need for pre-amplification or specialized recognition mechanisms. Based on these findings, we proposed the "Fragment Complementary Activation Strategy" (FCAS) and designed reverse fluorescence-enhanced lateral flow test strips (rFLTS) for the direct detection of miRNA-10b, achieving a limit of detection (LOD) of 5.53 fM and quantifying the miRNA-10b biomarker in clinical serum samples from glioma patients. Moreover, for the first time, we have developed the FCAS-based CRISPR/Cas12a system for miRNA in situ imaging, effectively recognizing tumor cells. The FCAS not only broadens the scope of CRISPR/Cas12a system target identification but also unlocks the potential for in-depth studies of CRISPR technology in many diagnostic settings. We proposed the 'Fragment Complementary Activation Strategy' (FCAS) based on the CRISPR/Cas12a system and designed fragment activators consisting of ssDNA and miRNA targets, enabling the direct detection of miRNAs.