CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics, but its utility in the sensing of microRNA (miRNA) biomarkers is limited by sensitivity, cost and robustness. Here, we describe a CRISPR-Dx method for the sensitive and cost-effective detection of miRNAs by rationally integrating CRISPR-Cas12a with DNA circuits. In this work, a modular catalytic hairpin assembly (CHA) circuit is designed to convert and amplify each target into multiple programmable DNA duplexes, which serve as triggers to initiate the trans-cleavage activity of CRISPR-Cas12a for further signal amplification. Such rational integration provides a generic assay for the effectively amplified detection of miRNA biomarkers. By simply tuning the variable regions in the CHA modules, this assay achieves sub-femtomolar sensitivity for different miRNA biomarkers, which improves the detection limit of CRISPR-Dx in the analysis of miRNA by 3–4 orders of magnitude. With the usage of the proposed assay, the sensitive assessment of miR-21 levels in different cancer cell lines and clinical serum samples has been achieved, providing a generic method for the sensitive detection of miRNA biomarkers in molecular diagnosis.

中文翻译：

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将CRISPR-Cas12a与DNA电路集成在一起，作为用于扩增检测microRNA的通用传感平台

基于CRISPR的诊断技术（CRISPR-Dx）在分子诊断中显示出了广阔的前景，但其在microRNA（miRNA）生物标志物检测中的实用性受到灵敏度，成本和耐用性的限制。在这里，我们描述了一种CRISPR-Dx方法，用于通过将CRISPR-Cas12a与DNA电路合理整合来灵敏且经济高效地检测miRNA。在这项工作中，设计了模块化催化发夹装配（CHA）电路，以将每个靶标转化并扩增为多个可编程的DNA双链体，从而启动反式CRISPR-Cas12a的切割活性，用于进一步的信号放大。这种合理的整合为有效扩增miRNA生物标记物提供了通用的检测方法。通过简单地调节CHA模块中的可变区，该测定可实现针对不同miRNA生物标记物的亚飞摩尔灵敏度，从而将CRISPR-Dx在miRNA分析中的检测限提高了3-4个数量级。通过使用拟议的测定方法，已实现了对不同癌细胞系和临床血清样品中miR-21水平的灵敏评估，为在分子诊断中灵敏检测miRNA生物标志物提供了一种通用方法。