Catalytic hairpin assembly-assisted split-T7 promoter-regulated CRISPR/Cas12a system for the sensitive analysis of microRNAs associated with coronary heart disease

Abstract

MicroRNAs (miRNAs) have emerged as promising non-invasive biomarkers for coronary heart disease (CHD); however, their accurate quantification remains challenging due to their short length, low abundance, and high sequence homology. Herein, we report a highly sensitive and specific biosensing platform by integrating catalytic hairpin assembly (CHA) with a split-T7 promoter-regulated CRISPR/Cas12a system. The key innovation lies in the split-T7 promoter architecture, which enables target-responsive reconstitution of a functional T7 promoter, driving robust in vitro transcription to generate abundant crRNA transcripts eliminating the need for pre-synthesized exogenous crRNA. This strategy achieves triple cascade amplification through three synergistic modules: CHA-mediated target recycling ensures efficient signal initiation; split-T7 promoter-driven transcription provides substantial signal enrichment; and CRISPR/Cas12a-based trans-cleavage delivers highly specific fluorescence readout. By eliminating pre-synthesized crRNA, the platform significantly reduces assay cost and complexity while maintaining excellent amplification efficiency. The method achieves a limit of detection as low as 38.9 aM for miRNA-155 within 90 min, exhibits single-base mismatch discrimination capability, and performs reliably in clinical specimens from CHD patients. With its modular design and robust performance, this cascade amplification platform offers a versatile and cost-effective tool for miRNA analysis, holding great promise for cardiovascular disease diagnosis and broader molecular diagnostics.