Rapid detection of Hepatitis C virus using CRISPR/Cas

项目摘要/摘要 2017年，世卫组织估计有7100万人患有慢性丙型肝炎病毒(丙型肝炎病毒)，但81%的活着患者 都不知道自己的感染状况。仅在2016年，世卫组织就报告了约39.9万人死于丙型肝炎病毒。 美国疾病控制与预防中心估计，2013-2016年间，美国约有210万人感染了丙型肝炎病毒，而只有 他们中的一小部分被正确诊断。对早期丙型肝炎病毒的快速可靠检测将使更快 并可明显降低死亡风险和感染率。创新的自检诊断平台 为了使用V型和VI型工程技术早期检测丙型肝炎病毒RNA，可以建立CRISPR/CAS系统。第V类和第VI类 当CRISPR/Cas系统与其特定的靶核酸序列结合时，激活次级侧枝核酸酶 能够以非特异性多次翻转的方式快速切割单链核酸的活性。通过检测 CRISPR/Cas系统的侧枝活性使用基于FRET的报告程序，最多10 nM的靶序列已经被 由杜德纳和张实验室荧光检测。通过使用逆转录酶和/或等温来预先扩增靶 DNA扩增，RNA或DNA的单分子检测已经实现，浓度低至凌晨2点在……里面 尚未发表的初步工作，CRISPR/Cas12a的工程CRISPR RNA(CrRNA)被发现放大了这一点 此外，还实现了高达400,000倍的灵敏度提高，检测极限为25 fM的PCA3 dsDNA 数小时，在30分钟内获得700 fM的HIV单链DNA，在30分钟内获得290 fM的丙型肝炎病毒单链DNA，而不需要目标预 放大。对CRISPR RNA的额外修饰提高了检测区分单个 点突变体。 根据初步数据，有以下具体目标。1)提高诊断的敏感性和特异性 通过将新的工程规则应用于CRISPR/Cas12a、CRISPR/Cas13a和 CRISPR/Cas14a系统，可以识别已知的病毒RNA拷贝，灵敏度为1毫升/毫升RNA 血。2)研制了一种100拷贝/毫升的纸基丙型肝炎病毒目标物肉眼比色检测装置 集中精神。3)使用优化的设备进行初步研究，以验证临床血液样本中的丙型肝炎病毒检测 来自健康、高危、感染和治疗的患者的准确率为95%。这种集成的方法将拥有所有 受保人定义的组件(负担得起、敏感、具体、用户友好、快速和坚固、无设备 和交付给最终用户)标准。这一快速诊断平台的开发将使更快 治疗，减少暴发和患者更快的反应。在未来，这种方法将能够检测到联合感染 包括结核病、人乳头瘤病毒和艾滋病毒，以及乙肝病毒，这些都是丙型肝炎病毒感染者死亡的主要原因。

PROJECT ABSTRACT/SUMMARY In 2017, WHO estimated 71 million people had chronic Hepatitis C Virus (HCV) but 81% 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 2.1 million people were infected with HCV within the US and only a fraction of them were diagnosed properly. A rapid and reliable detection of an early-stage HCV would allow quicker intervention and can significantly reduce the risk of death and infection rate. An innovative self-testing diagnostic platform for early detection of HCV RNA using engineered type V and VI CRISPR/Cas systems can be created. 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. By detecting the collateral activity of CRISPR/Cas systems using a FRET-based reporter, up to 10 nM of target sequence has been fluorescently detected by Doudna and Zhang labs. By pre-amplifying a target using a reverse transcriptase and/or isothermal DNA amplification, single molecule detection of RNA or DNA has been achieved with concentrations as low as 2 aM. In preliminary unpublished work, engineered CRISPR RNA (crRNA) for CRISPR/Cas12a were discovered to amplify this further and achieved up to > 400,000-fold improved sensitivity with the limit of detection of 25 fM of PCA3 dsDNA in 6 hours, 700 fM of HIV ssDNA in 30 minutes, and 290 fM HCV ssDNA in 30 minutes without requiring target pre- amplification. Additional modifications on the CRISPR RNA improved the specificity of detection discriminating single point mutants. Based on the preliminary data, there are following specific goals. 1) To enhance sensitivity and specificity of CRISPR/Cas systems by applying novel engineering rules to different orthologs of CRISPR/Cas12a, CRISPR/Cas13a, and CRISPR/Cas14a systems that can identify known viral RNA copies with a sensitivity of 100 copies of RNA in 1 mL of blood. 2) To develop a paper-based device for colorimetric detection of a HCV target by naked eye at 100 target copies/mL concentration. 3) To perform a pilot study with the optimized device for validating HCV detection in clinical blood samples from healthy, high-risk, infected and treated patients with 95% accuracy. This integrated approach will have 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 this rapid diagnostic platform would allow quicker treatment, reduce outbreak and faster response from patients. In future, this approach would enable detection of co-infections including TB, HPV, and HIV, and HBV that are the major causes of death in HCV-infected populations.