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

点击翻译按钮获取中文摘要

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.