Rapid detection of Hepatitis C virus using CRISPR/Cas

项目摘要/总结

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 In preliminary unpublished work, engineered CRISPR RNA (crRNA) for CRISPR/Cas12a were discovered to amplify this further and achieved up to 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