The target-dependent endonuclease activity (also known as the trans-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.

中文翻译：

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CRISPR-Cas 驱动的单微电机 (Cas-DSM) 可在单分子水平上直接检测核酸生物标志物

靶标依赖性核酸内切酶活性（也称为反式-裂解活性）的 CRISPR-Cas 系统激发了人们对开发用于核酸诊断的新生传感策略的极大兴趣。尽管进行了多次尝试，但大多数敏感的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测试时间，以及引入假阳性的风险结果。在这项工作中，我们提出了 CRISPR-Cas-Driven Single Micromotor (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，可用于定量检测各种复杂临床样本以及单个细胞中的 DNA/RNA，无需任何预扩增过程。由于CRISPR/Cas系统的优良特性，包括恒温、设计简单、特异性高和灵活的可编程性，Cas-DSM可以作为核酸检测的简单通用平台。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。