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<p>Accurate and sensitive detection of low‑abundance microRNAs (miRNAs) is critical for the early diagnosis of liver cirrhosis, yet remains challenging due to limitations in the sensitivity, specificity, and operational complexity of conventional methods. Here, we report a novel fluorescent biosensor based on a circular CRISPR RNA (crRNA)‑modulated CRISPR/Cas12a system for the detection of liver cirrhosis ‑associated let‑7a. A key innovation of this design is the implementation of a circular crRNA, which demonstrates markedly enhanced stability against enzymatic degradation compared with its linear counterpart, thereby conferring greater assay robustness and interference resistance, an essential advantage for clinical translation. The assay integrates catalytic hairpin assembly (CHA) and RNA‑cleaving DNAzymes to convert target recognition into activation of Cas12a trans‑cleavage activity. This biosensor achieves an exceptionally low detection limit of 105 aM and a broad linear range from 0.5 fM to 50 pM, while exhibiting high specificity and reproducibility (RSD &lt; 5%). Notably, it maintains consistent performance in complex media (10% human serum) and reliably quantifies let‑7a levels in clinical blood samples. The results show strong agreement with standard RT‑qPCR measurements, enabling clear discrimination between liver cirrhosis patients and healthy individuals. This work not only presents a highly sensitive and robust sensing platform but also confirms its substantial clinical potential for the early detection of miRNA‑related diseases.</p> Graphical Abstract <p></p>

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Catalytic hairpin assembly of trivalent RNA-cleaving DNAzyme junctions regulates circular crRNA for sensitive and accurate CRISPR/Cas12a based liver cirrhosis related MicroRNA analysis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月13日

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  • Yongle Zheng,
  • Xiaoming Wang,
  • Meiyan Xu,
  • Hui Zhao

摘要

Accurate and sensitive detection of low‑abundance microRNAs (miRNAs) is critical for the early diagnosis of liver cirrhosis, yet remains challenging due to limitations in the sensitivity, specificity, and operational complexity of conventional methods. Here, we report a novel fluorescent biosensor based on a circular CRISPR RNA (crRNA)‑modulated CRISPR/Cas12a system for the detection of liver cirrhosis ‑associated let‑7a. A key innovation of this design is the implementation of a circular crRNA, which demonstrates markedly enhanced stability against enzymatic degradation compared with its linear counterpart, thereby conferring greater assay robustness and interference resistance, an essential advantage for clinical translation. The assay integrates catalytic hairpin assembly (CHA) and RNA‑cleaving DNAzymes to convert target recognition into activation of Cas12a trans‑cleavage activity. This biosensor achieves an exceptionally low detection limit of 105 aM and a broad linear range from 0.5 fM to 50 pM, while exhibiting high specificity and reproducibility (RSD < 5%). Notably, it maintains consistent performance in complex media (10% human serum) and reliably quantifies let‑7a levels in clinical blood samples. The results show strong agreement with standard RT‑qPCR measurements, enabling clear discrimination between liver cirrhosis patients and healthy individuals. This work not only presents a highly sensitive and robust sensing platform but also confirms its substantial clinical potential for the early detection of miRNA‑related diseases.

Graphical Abstract