Rapid point-of-care detection of Hepatitis C viral RNA using multiplexed CRISPR/Cas platforms

项目摘要/总结 2017年，世卫组织估计有7100万人患有慢性丙型肝炎病毒（HCV），但40-50%的人 患者不知道自己的感染状况。仅在2016年，估计有399，000例HCV相关死亡， 据WHO报道。CDC估计，在2013年至2016年期间，约有240万人感染了HCV 在美国，只有一小部分人被正确诊断。一种快速廉价的HCV检测方法 RNA将允许更快的干预，并可以显着降低死亡风险和感染率，特别是 在资源有限的环境中。通过以独特的方式工程化和多路复用CRISPR/Cas系统， 可以在30分钟内对血液样品实现HCV RNA低拷贝的灵敏检测。这个项目 提出了两种高度创新的基于CRISPR的方法的开发和临床验证， 侧流试验中的HCV基因型。V型和VI型CRISPR/Cas系统与其特异性靶标结合时 核酸序列，激活次级侧链核酸酶活性，其可以快速切割单链 核酸以非特异性多重转换方式。这种附带核酸酶活性已被用于 快速检测核酸然而，它们具有纳摩尔的灵敏度，并且需要靶的预扩增 以实现临床应用所需的阿托摩尔检测。虽然预放大可以通过以下方式实现： 等温技术，这需要额外的操作步骤和稳定的温度控制， 分析的时间和成本。 为了消除对靶预扩增的需要，同时保持高灵敏度和特异性， 高风险/高回报项目提出了两种创新方法来实现HCV RNA的快速检测 而没有任何靶扩增。对于第一个目标，最近开发的“CRISPR-ENHANCE”（CE）平台， PI的实验室在30分钟内实现了毫微微摩尔核酸检测，而无需任何靶向预扩增 （Nguyen等人，国家通讯社，2020年）将以组合方式进行测试，以进一步提高对 使用侧流测定法检测HCV RNA的临床水平。第二个目标是发展和临床 验证基于CRISPR链反应（CCR）的检测方法，使用多重PCR技术检测HCV RNA和基因型 侧流测定和使用简单的基于荧光的即时装置定量RNA。两者 方法将在急性/慢性感染的库存样本中进行临床验证， 与丙型肝炎治疗登记处合作监测接受抗病毒治疗的患者， 研究网络（HCV-TARGET），一个由领先的HCV研究人员组成的国际联盟。所有的 组件定义的保证（负担得起的，敏感的，具体的，用户友好的，快速和强大的， 无需设备和可向最终用户提供）的标准。快速诊断平台的开发 将允许更快的治疗、减少疫情爆发和患者更快的反应。今后，这种方法将 能够检测HCV的其他基因型和病原体。

PROJECT ABSTRACT/SUMMARY In 2017, WHO estimated 71 million people had chronic Hepatitis C Virus (HCV) but 40-50% of the living patients were unaware of their infection status. In 2016 alone, an estimated 399,000 HCV-related deaths were reported by WHO. CDC estimates that between 2013-2016, around 2.4 million people were infected with HCV within the US and only a fraction of them were diagnosed properly. A rapid and inexpensive detection of HCV RNA would allow quicker intervention and can significantly reduce the risk of death and infection rate, especially in resource-limited settings. By engineering and multiplexing CRISPR/Cas systems in unique ways ultra- sensitive detection of low copies of HCV RNA can be achieved for blood samples within 30 minutes. This project proposes development and clinical validation of two highly innovative CRISPR-based approaches for detecting HCV genotype in a lateral flow assay. Type V and VI CRISPR/Cas systems when bound with their specific target nucleic acid sequence, activate a secondary collateral nuclease activity that can rapidly cleave single-stranded nucleic acids in a non-specific multiple turnover manner. This collateral nuclease activity has been utilized for rapidly detecting nucleic acids. However, they have nanomolar sensitivity and require pre-amplification of a target to achieve attomolar detection that is desirable for clinical use. While pre-amplification can be achieved by isothermal techniques this requires additional manipulation steps and a stable temperature control increasing the time and cost of an assay. To eliminate the need for target pre-amplification while maintaining high sensitivity and specificity, this high-risk/high-reward project proposes two innovative approaches to achieve rapid detection of HCV RNA without any target amplification. For the first aim, a recently developed `CRISPR-ENHANCE' (CE) platform from the PI's lab that achieved femtomolar detection of nucleic acids in 30 minutes without any target pre-amplification (Nguyen et al., Nat. Comm., 2020) will be tested in a combinatorial fashion to further enhance the sensitivity for detecting clinical levels of HCV RNA using a lateral flow assay. The second aim is to develop and clinically validate a CRISPR Chain Reaction (CCR) based test for detecting HCV RNA and genotypes using multiplexed lateral flow assay and quantifying RNA using a simple fluorescence-based point-of-care device. Both the approaches will be clinically validated in banked samples with acute/chronic infections as well as longitudinally monitor patients undergoing anti-viral therapy in collaboration with Hepatitis C Therapeutic Registry and Research Network (HCV-TARGET), an international consortium of leading HCV investigators. All the components as defined by the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) criteria by WHO. The development of a rapid diagnostic platform would allow quicker treatment, reduce outbreak and faster response from patients. In future, this approach would enable detection of other genotypes of HCV and etiologic agents.