Benchtop to home : development of amplicon-eliminating CRISPR-based at-home diagnostic platforms

Abstract

The timely detection and management of infectious diseases requires sensitive, accurate, and reliable detection of nucleic acid (DNA) based biomarkers due to the ease of target amplification comparing to other antigen-based biomarkers. With well-characterized DNA activated enzymes, one can develop a DNA sensing platform easily with high analytical result in clinical use or even home use. Nevertheless, to linkage a lab-setting molecular assay to home-settings, the professionals handling and environmental control are two main limits. Thus, the breaking through strategy to bridge the lab-to-home technology translation demands a well regulation in one-pot reaction to keep the good performance at-home including sensitivity and accuracy. This thesis showcases two applications of CRISPR/Cas as a regulation tool to transduce the amplified DNA amplicons to signal readout with a clear product pollution from amplicon-derived aerosol and PCR level sensitivity. The first project in chapter 2 focuses on the compatibility of CRISPR-based amplicon digestion with target amplification in one-pot reaction at one temperature without compromising the kinetics. We demonstrated that by employing a CRISPR-based contamination-free strategy to site activator sequence within the specific primer-binding region that is crucial in the target-independent exponential amplification stage after the target-dependent initiation stage, the optimized reaction can eliminate the amplicon products and detect sub attomolar quantities of DNA target within 30 minutes. The second project in chapter 3 utilizes recombinase enzymes in the loop-mediated amplification to explore the universality of the amplicon-eliminating strategy with simpler primer design from two sets of primer to one set. Chapter 4 illustrates a wearable biosensor designed suitable for the translation of the above-mentioned molecular assay to easy-to-handle self-testing assay at home. We first stablished with a colorimetric cascade coated by DNA hydrogel network for in-situ real-time visual readout with DNA detection reaction spontaneously; then, we characterized its ability to eliminate amplicon-derived false positive in at-home settings by detecting both DNA and RNA viral pathogens in several currently popular viral infectious diseases with no closed system to take control of the contamination. Lastly, this dissertation concludes a summary on further work to be done soon to push these technologies to enter the real market. In particular, after the SARS-CoV-2 pandemic and the other seasonal influenzas affecting billions of lives globally, we offer thermostatic diagnostic reactions without amplicon aerosol contamination that are expected to become a breakthrough technology for molecular diagnostics to enter the era of self-detection because it is free from equipment dependence and environmental restrictions.

Date of Award	2024
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	I-ming HSING (Supervisor)