Peng Shuang, Tan Zhen, Chen Siyu, Lei Chunyang, Nie Zhou
State Key Laboratory of Chemo/Biosensing and Chemometrics , College of Chemistry and Chemical Engineering , Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology , Hunan University , Changsha 410082 , P. R. China . Email:
Chem Sci. 2020 Jun 19;11(28):7362-7368. doi: 10.1039/d0sc03084h. eCollection 2020 Jul 28.
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 -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.
基于CRISPR的诊断技术（CRISPR-Dx）在分子诊断领域展现出了巨大的潜力，但其在检测微小RNA（miRNA）生物标志物方面的实用性受到灵敏度、成本和稳健性的限制。在此，我们描述了一种通过合理整合CRISPR-Cas12a与DNA电路来灵敏且经济高效地检测miRNA的CRISPR-Dx方法。在这项工作中，设计了一种模块化催化发夹组装（CHA）电路，将每个靶标转化并扩增为多个可编程的DNA双链体，这些双链体作为触发物启动CRISPR-Cas12a的切割活性以进行进一步的信号放大。这种合理整合为有效扩增检测miRNA生物标志物提供了一种通用检测方法。通过简单调整CHA模块中的可变区域，该检测方法对不同的miRNA生物标志物实现了亚飞摩尔级别的灵敏度，将CRISPR-Dx在miRNA分析中的检测限提高了3至4个数量级。使用所提出的检测方法，已实现了对不同癌细胞系和临床血清样本中miR-21水平的灵敏评估，为分子诊断中灵敏检测miRNA生物标志物提供了一种通用方法。
