Meng Tao, Kang Qi, Xu Jiashuo, Zhao Shuang, Liu Tianqi, Zhou Dianming, Gong Xiaoqun, Zhang Jianyu
School of Pharmaceutical Science and Technology, Faculty of Medicine, Tianjin University, Tianjin, 300072, PR China.
Department of Cardiology, Tianjin Medical University General Hospital, Tianjin, 300041, PR China.
Talanta. 2025 Oct 1;293:128061. doi: 10.1016/j.talanta.2025.128061. Epub 2025 Apr 5.
The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans-cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10 U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.
CRISPR/Cas12a系统因其特异的靶标识别能力和高效的反式切割活性，已成为生物传感领域的强大工具。然而，CRISPR/Cas12a系统直接用于痕量检测时面临的一个问题是单循环消化的线性扩增效率。在此，我们提出了一种新型的发夹报告基因驱动的CRISPR/Cas12a（HR-CRISPR）扩增系统，该系统在CRISPR/Cas12a平台内建立了一个正反馈回路，以在一步反应中完成指数级且灵敏的信号放大。作为概念验证，我们将此策略应用于无需预扩增程序的末端脱氧核苷酸转移酶（TdT）活性检测。TdT延伸的polyT链与crRNA杂交，激活Cas12a，然后Cas12a切割FQ-发夹报告基因。切割产物以crRNA为模板通过逆转录酶进一步延伸，重新激活Cas12a并产生指数级放大的荧光信号。该检测方法为定量TdT活性提供了一种简单但高度灵敏的方法，检测下限低至4.55×10 U。此外，它适用于抑制剂筛选以及监测人血清样本中的TdT活性。
