Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes’ RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.

中文翻译：

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用于 SARS-CoV-2 诊断的新型微型 CRISPR-Cas13 系统

快速的即时护理 (POC) 诊断对于减轻当前（和未来）流行病的影响至关重要；然而，目前检测严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的方法需要复杂的实验室测试，这些测试通常在场外进行，并且需要大量时间。CRISPR-Cas系统已被用来开发用于核酸检测的敏感且特异的平台。这些检测平台利用 CRISPR 酶对 RNA 和 DNA 靶标的 RNA 引导特异性以及单链 RNA 和 DNA 报告基因的附带反式活性。微生物基因组拥有广泛的 CRISPR 酶，具有不同的特异性和附带活性水平；鉴定新酶可能会改善基于 CRISPR 的诊断。在这里，我们鉴定了一种新的 Cas13 变体，将其命名为微型 Cas13 (mCas13)，并表征了其催化活性。然后，我们利用该系统设计、构建和测试将逆转录环介导等温扩增 (RT-LAMP) 与 mCas13 系统相结合的 SARS-CoV-2 检测模块，以检测合成样本和临床样本中的 SARS-CoV-2。我们的系统表现出与其他 CRISPR 系统相当的灵敏度和特异性。这项工作扩展了 Cas13 酶在诊断和潜在体内应用（包括 RNA 敲低和编辑）中的功能和应用。重要的是，我们的系统可以潜在地适应并用于多种病原体的大规模测试，包括 RNA 和 DNA 病毒以及细菌。