Exonuclease-III Assisted Signal Cycle Integrating with Self-Priming Mediated Chain Extension for Sensitive and Reliable MicroRNA Detection

MicroRNA (miRNA) is pivotal in regulating pathological progression and may serve as a significant biomarker for early diagnosis, treatment, and management strategies for atherosclerosis. This study produced a self-priming amplification-accelerated CRISPR/Cas system-based method for the sensitive and selective detection of miRNA by merging Exo-III-assisted target recycling, self-priming-mediated chain extension, and the CRISPR/Cas12a system. The sensor comprises three stages: (i) the creation of a substrate template via Exo-III mediated target recycling and DNA ligase assisted ligation; (ii) the exponential isothermal reaction facilitated by DNA polymerase for signal amplification; (iii) the trans-cleavage activity of CRISPR/Cas12a after recognizing the amplification product generates signals. We employed miRNA-21 as a target. The strategy enables sensitive detection of miRNA-21 without the use of primers, and the unique design of the CRISPR/sgRNA complex efficiently mitigates background signal interference. The sensor can recognize single-base mutant homologous sequences and demonstrate a steady performance in complicated biological matrices. This sensor has been effectively employed to precisely assess miRNA-21 in engineered clinical samples, showcasing its significant potential in clinical diagnostics and of atherosclerosis.