Orthogonal Cas13a/Cas12a cascade for one-pot amplification-free detection of miRNA-21

MicroRNAs (miRNAs) are promising biomarkers for clinical diagnosis and disease monitoring. However, current CRISPR/Cas-based miRNA sensors generally require reverse transcription or nucleic acid amplification to improve sensitivity, which complicates the workflow and increases the risk of contamination, nonspecific amplification, and false-positive results. Herein, we developed a one pot amplification-free Cas13a/Cas12a cascade platform based on a designed dual-functional molecular bridge probe, Conv HP-3, which served as both a substrate for Cas13a-mediated trans-cleavage and an activator for Cas12a-mediated trans-cleavage, thereby linking target recognition to cascade signal amplification for miRNA-21 detection. Following the introduction of miRNA-21, Cas13a was specifically activated through target-crRNA recognition and cleaved the Conv HP-3 probe to release a Cas12a-activating DNA fragment. This fragment subsequently triggered Cas12a-mediated cleavage of the ssDNA reporter, generating a markedly enhanced fluorescence signal. This one-pot Cas13a/Cas12a cascade fluorescence biosensor enabled quantitative detection of miRNA-21 over a concentration range of 1-1000 pmol/L within 60 min, with a low detection limit of 0.66 pM. Notably, the assay achieved average recoveries ranging from 95.97% to 108.59%, with a variation between 0.6% and 1.78%, demonstrating its good accuracy and precision. This biosensing platform shows great promise for the rapid and sensitive detection of miRNAs in clinical applications.