Isothermal miRNA Detection via Ligation-Dependent CRISPR/Cas13a Activation

Abstract

Here, we present a rapid isothermal strategy for miRNA detection based on ligation-dependent generation of Cas13a-activating RNA and CRISPR/Cas13a-mediated signal amplification. The system employs two DNA probes: a hairpin-structured probe containing a double-stranded T7 promoter within its stem and a second probe encoding a template for Cas13a activator RNA. In the presence of a target miRNA, the probes are ligated to form a functional transcription template, enabling the generation of Cas13a-activating RNA. The resulting RNA activates the Cas13a–crRNA complex, inducing collateral cleavage of a fluorescent reporter for signal generation. The method achieves a limit of detection of 2.56 pM for miR-21 and 11.64 pM for miR-10a, with a linear response over 0–500 pM, while maintaining high specificity with negligible responses to nontarget miRNAs. Importantly, the entire assay is completed within 40 min at 37 °C, highlighting its rapid detection capability. The applicability of the platform is validated using miRNA extracted from HepG2, HeLa, and MCF-7 cells, yielding results consistent with RT-qPCR analysis. Owing to its short assay time, operation at 37 °C, and sequence programmability through probe redesign, this strategy provides a useful framework for miRNA detection.