Marpaung, David Septian Sumanto (57209025328); Yap Sinaga, Ayu Oshin (60349849900); Damayanti, Damayanti (57200040345); Utari, Ni Wayan Arya (60507634800); Harmiansyah (58487516100); Karangan, Arthur (57223864368); Kusmali, Muh (57218864569)
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. © 2026 Wiley-VCH GmbH.
Department of Biosystems Engineering, Institut Teknologi Sumatera, Lampung, Lampung Selatan, Indonesia; Department of Biology, Institut Teknologi Sumatera, Lampung, Lampung Selatan, Indonesia; Department of Chemical Engineering, Institut Teknologi Sumatera, Lampung, Lampung Selatan, Indonesia; Research and Development Department, PT. Mitra Prodin, Bali, Kecamatan Sukawati, Indonesia
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