Department of Immunology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Department of Biological Sciences, Dietrich School of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
J Med Virol. 2021 Jul;93(7):4198-4204. doi: 10.1002/jmv.26889. Epub 2021 Mar 25.
Nucleic acid detection is a necessary part of medical treatment and fieldwork. However, the current detection technologies are far from ideal. A lack of timely and accessible testing for identifying cases and close contacts has allowed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative virus of the ongoing coronavirus disease-2019 (COVID-19) pandemic, to spread uncontrollably. The slow and expensive detection of mutations-predictors for chronic diseases such as cancer-form a barrier to personalized treatment. A recently developed diagnostic assay is ideal and field-ready-it relies on CRISPR-Cas13. CRISPR-Cas13 works similarly to other CRISPR systems: Cas13 is guided by a crRNA to cleave next to a specific RNA target sequence. Additionally, Cas13 boasts a unique collateral cleavage activity; collateral cleavage of a fluorescent reporter detects the presence of the target sequence in sample RNA. This system forms the basis of CRISPR-Cas13 diagnostic assays. CRISPR-Cas13 assays have >95% sensitivity and >99% specificity. Detection is rapid (<2 h), inexpensive ($0.05 per test), and portable-a test using lateral flow strips is akin to a pregnancy test. The recent adaptation of micro-well chips facilitates high-level multiplexing and is high-throughput. In this review, we cover the development of CRISPR-Cas13 assays for medical diagnosis, discuss the advantages of CRISPR-Cas13-based diagnosis over the traditional reverse transcription polymerase chain reaction (RT-PCR), and present examples of detection from real patient samples.
核酸检测是医疗和现场工作的必要组成部分。然而，目前的检测技术远非理想。缺乏及时和可获得的检测来识别病例和密切接触者，使得严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2）——导致当前 2019 年冠状病毒病（COVID-19）大流行的致病病毒——无法得到控制地传播。对突变的缓慢和昂贵的检测——如癌症等慢性病的预测因子——成为个性化治疗的障碍。最近开发的诊断检测方法非常理想且适合现场使用——它依赖于 CRISPR-Cas13。CRISPR-Cas13 的工作原理类似于其他 CRISPR 系统：Cas13 由 crRNA 引导，在特定 RNA 靶序列的旁边切割。此外，Cas13 具有独特的旁切活性；荧光报告的旁切检测到样品 RNA 中靶序列的存在。该系统构成了 CRISPR-Cas13 诊断检测的基础。CRISPR-Cas13 检测方法的灵敏度>95%，特异性>99%。检测速度快（<2 小时），价格低廉（每个测试 0.05 美元），且便携——使用侧流条的测试类似于妊娠测试。最近对微井芯片的适应促进了高水平的多重检测和高通量。在这篇综述中，我们介绍了用于医疗诊断的 CRISPR-Cas13 检测方法的发展，讨论了基于 CRISPR-Cas13 的诊断相对于传统逆转录聚合酶链反应（RT-PCR）的优势，并展示了来自真实患者样本的检测示例。
