SPRINT-Cas12a: A Single Leading-Hairpin Probe-Enabled Dual-Pathway Activation Strategy for Sensitive miRNA Detection

Abstract CRISPR−Cas12a possesses robust trans-cleavage activity and has emerged as a powerful tool for nucleic acid detection. However, the efficient integration of Cas12a trans-cleavage with direct miRNA recognition and signal amplification remains challenging, particularly because conventional assay designs often rely on multiple probes or a single activation pathway. Here, we developed SPRINT-Cas12a, a single-probe RNA-triggered integrated trans-cleavage platform for sensitive miRNA detection. In this design, a leading hairpin probe (L-HP) specifically recognizes the target miRNA and initiates polymerase-mediated cascade isothermal amplification, synchronously generating two structurally distinct Cas12a-activating products: an RNA−DNA complex and a long-chain micro-hairpin probe (L-MHP). These products provide complementary activation pathways for Cas12a, thereby promoting reporter cleavage and fluorescence signal generation. The single-probe architecture simplifies probe design while enabling synergistic signal amplification through dual-pathway Cas12a activation. The platform was successfully adapted for the detection of miR-21 and miR-122, two miRNAs with distinct sequences, demonstrating its modularity and sequence versatility. Using miR-192 as a hepatocellular carcinoma-associated target, the method achieved sensitive detection in serum samples and distinguished patients with hepatocellular carcinoma from healthy controls, with results consistent with stem-loop quantitative reverse-transcription PCR. In addition, the platform was coupled with a lateral flow assay to provide an instrument-free visual readout. Depending on the amplification and readout format, the assay could be completed within 90 min to 120 min using simple laboratory equipment. Overall, SPRINT-Cas12a provides a single-probe, dual-pathway strategy for Cas12a-mediated miRNA detection and shows promise for portable molecular diagnostics and point-of-care testing.