Institute for Integrative Systems Biology (I2SysBio), CSIC - University of Valencia, 46980 Paterna, Spain.
Microbiology Service, Clinic University Hospital, INCLIVA Biomedical Research Institute, 46010 Valencia, Spain.
Anal Chem. 2023 Jun 27;95(25):9564-9574. doi: 10.1021/acs.analchem.3c01041. Epub 2023 May 19.
Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.
由不同病毒引起的传染病爆发，包括新型呼吸道病毒 SARS-CoV-2，正在对我们的全球社会造成挑战；因此，多功能的病毒检测方法将能够进行有计划和更快的反应。在这里，我们提出了一种基于 CRISPR-Cas9 的新型核酸检测策略，其作用模式依赖于链置换而不是旁催化，使用 Cas9 核酸酶。在预扩增过程中，合适的分子信标在靶向时与三元 CRISPR 复合物相互作用，产生荧光信号。我们表明，可以用 CRISPR-Cas9 检测来自患者样本的 SARS-CoV-2 DNA 扩增子。我们还表明，CRISPR-Cas9 允许使用相同的核酸酶同时检测不同的 DNA 扩增子，无论是检测不同的 SARS-CoV-2 区域还是不同的呼吸道病毒。此外，我们证明了工程 DNA 逻辑电路可以处理 CRISPR 复合物检测到的不同 SARS-CoV-2 信号。总之，这种用于分子信标开启的 CRISPR-Cas9 R 环使用（COLUMBO）平台允许在单个管中进行多重检测，补充了现有的基于 CRISPR 的方法，并显示了诊断和生物计算的潜力。
