Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306, USA.
Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, 40530, Sweden.
Nat Commun. 2021 Sep 27;12(1):5653. doi: 10.1038/s41467-021-25977-7.
Among the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.
在现有的病毒检测方法中，基于可编程的 CRISPR-Cas 酶的方法具有快速报告和高灵敏度的优势，而不需要热循环仪。III-A 型 CRISPR-Cas 系统是一种多组分、多方面的免疫效应器，由病毒 RNA 激活，以前由于酶的重新组装过程和功能复杂，尚未被重新用于疾病检测。在这里，我们描述了一种基于体内重建的 III-A 型 CRISPR-Cas 系统的病毒检测方法的构建和应用。该系统利用 RNA 和转录激活的双重核酸切割活性以及内部信号放大，允许在多个设置下进行高灵敏度的病毒检测。我们展示了该 III-A 型系统方法在检测 SARS-CoV-2 中的应用，该方法在无扩增的情况下达到了 2000 拷贝/μl 的灵敏度，在等温扩增的情况下达到了 60 拷贝/μl 的灵敏度，并且可以在这两种情况下诊断 SARS-CoV-2 感染的患者。高灵敏度、灵活的反应条件和小分子驱动的扩增使 III-A 型系统成为一种具有广泛应用前景的独特的核酸检测方法。
