Rapid detection of Hepatitis C virus using CRISPR/Cas

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81%的存活患者 他们不知道自己的感染状况。仅在2016年，世卫组织就报告了约399，000例HCV相关死亡。 CDC估计，2013 - 2016年间，美国约有210万人感染HCV， 他们中的一部分人得到了正确的诊断。快速可靠地检测早期HCV将使更快地 干预，可以显着降低死亡风险和感染率。创新的自我检测诊断平台 用于使用工程化的V型和VI型CRISPR/Cas系统早期检测HCV RNA。类型V和VI 当CRISPR/Cas系统与其特异性靶核酸序列结合时，其激活次级附带核酸酶 可以以非特异性多重转换方式快速切割单链核酸的活性。通过检测 使用基于FRET的报告基因的CRISPR/Cas系统的并行活性，已经将高达10 nM的靶序列 由Doudna和Zhang实验室荧光检测。通过使用逆转录酶和/或等温扩增来预扩增靶标， DNA扩增、RNA或DNA的单分子检测已经在低至2aM的浓度下实现。在 初步未发表的工作，CRISPR/Cas12a的工程化CRISPR RNA（crRNA）被发现可以扩增这种 进一步实现了高达> 400，000倍的灵敏度提高，检测限为25 fM的PCA3 dsDNA在6 小时，700 fM的HIV ssDNA在30分钟内，290 fM的HCV ssDNA在30分钟内，而不需要靶前处理。 放大对CRISPR RNA的额外修饰提高了检测区分单个DNA的特异性。 点突变体 根据初步数据，有以下具体目标。1)为了提高灵敏度和特异性， 通过将新的工程化规则应用于CRISPR/Cas12a、CRISPR/Cas13a和CRISPR/Cas13b的不同直系同源物， CRISPR/Cas14a系统可以识别已知的病毒RNA拷贝，灵敏度为100个拷贝的RNA在1 mL的 血2)开发一种用于以100靶拷贝/mL通过肉眼比色检测HCV靶的纸基装置 浓度. 3)使用优化的设备进行试点研究，以验证临床血液样本中的HCV检测 从健康的、高风险的、感染的和接受治疗的患者中，准确率为95%。这一综合办法将包括所有 ASSURED定义的组件（经济实惠、灵敏、具体、用户友好、快速和可靠、无需设备 并可向最终用户提供）的标准。这种快速诊断平台的开发将使人们能够更快地 治疗，减少爆发和更快的反应，从病人。在未来，这种方法将能够检测到合并感染 包括TB、HPV和HIV以及HBV，它们是HCV感染人群的主要死亡原因。

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 In preliminary unpublished work, engineered CRISPR RNA (crRNA) for CRISPR/Cas12a were discovered to amplify this further and achieved up to 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.