Integration of CRISPR/Cas12a and Toehold-Mediated Strand Displacement for Alternative Conventional miRNA Detection

MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression and serve as powerful biomarkers for cancer and other diseases. Conventional detection methods such as RT-qPCR, Northern blotting, microarrays, and next-generation sequencing provide robust analytical capabilities but remain limited by complexity, cost, and poor suitability for point-of-care diagnostics. CRISPR/Cas12a has emerged as a versatile nucleic acid detection platform with high specificity and sensitivity. However, its intrinsic preference for DNA substrates restricts direct application to miRNA sensing. Early CRISPR/Cas12a-based assays relied on enzymatic amplification, direct RNA-induced activation, or split-component designs, each offering proof-of-concept feasibility but facing trade-offs in sensitivity, workflow complexity, or robustness. Toehold-mediated strand displacement (TSD) provides a powerful alternative by converting miRNA inputs into DNA activators or crRNAs that efficiently trigger Cas12a. This integration enables enzyme-free amplification, programmable logic operations, and enhanced sensitivity, while reducing reliance on multienzyme cascades. This review critically evaluates conventional, enzymatic, direct, and split-based CRISPR/Cas12a strategies and emphasizes emerging TSD-assisted platforms as next-generation solutions for sensitive, specific, and portable miRNA detection.