Chen Desheng, Liang Yuanwen, Wang Honghong, Wang Hui, Su Fengxia, Zhang Pengbo, Wang Shuhui, Liu Weiliang, Li Zhengping
Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, China.
Anal Chem. 2023 Apr 4;95(13):5729-5737. doi: 10.1021/acs.analchem.2c05767. Epub 2023 Mar 21.
The target-dependent endonuclease activity (also known as the -cleavage activity) of CRISPR-Cas systems has stimulated great interest in the development of nascent sensing strategies for nucleic acid diagnostics. Despite many attempts, the majority of the sensitive CRISPR-Cas diagnostics strategies mainly rely on nucleic acid preamplification, which generally needs complex probes/primers designs, multiple experimental steps, and a longer testing time, as well as introducing the risk of false-positive results. In this work, we propose the CRISPR-Cas-Driven Single Micromotor (Cas-DSM), which can directly detect the nucleic acid targets at a single-molecule level with high specificity. We have demonstrated that the Cas-DSM is a reliable and practical method for the quantitative detection of DNA/RNA in various complex clinical samples as well as in individual cells without any preamplification processes. Due to the excellent features of the CRISPR/Cas system, including constant temperature, simple design, high specificity, and flexible programmability, the Cas-DSM could serve as a simple and universal platform for nucleic acid detection. More importantly, this work will provide a breakthrough for the development of next-generation amplification-free CRISPR/Cas sensing toolboxes.
CRISPR-Cas系统的靶标依赖性内切核酸酶活性（也称为切割活性）激发了人们对开发用于核酸诊断的新型传感策略的浓厚兴趣。尽管进行了许多尝试，但大多数灵敏的CRISPR-Cas诊断策略主要依赖于核酸预扩增，这通常需要复杂的探针/引物设计、多个实验步骤和更长的检测时间，还会引入假阳性结果的风险。在这项工作中，我们提出了CRISPR-Cas驱动的单微马达（Cas-DSM），它可以在单分子水平上以高特异性直接检测核酸靶标。我们已经证明，Cas-DSM是一种可靠且实用的方法，可用于在无需任何预扩增过程的情况下对各种复杂临床样本以及单个细胞中的DNA/RNA进行定量检测。由于CRISPR/Cas系统具有恒温、设计简单、特异性高和可编程性灵活等优异特性，Cas-DSM可作为一个简单通用的核酸检测平台。更重要的是，这项工作将为下一代无扩增CRISPR/Cas传感工具箱的开发提供突破。
