Amplification strategies for CRISPR-based diagnostics
File(s)
Author(s)
Christopher, Adrianus
Type
Thesis
Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics are currently emerging nucleic acid detection technologies. They have the potential to be developed into point-of-care (PoC) diagnostic platforms to be applicable in low-resource settings for the accurate detection of a wide range of diseases. However, state-of-the-art CRISPR platforms suffer from low sensitivity in PoC settings. To address this limitation, this thesis presents the development of two amplification strategies for CRISPR-based diagnostics: (i) a nanozyme-catalyzed immunosorbent assay (NLISA) with colorimetric readout and (ii) a polymerization-based amplification with turbidity readout. (i) NLISA was combined with a system comprised of CRISPR/Cas13 to generate a quantitative diagnostic platform (CrisprZyme) capable of detecting different types of non-coding RNA: microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA). Whilst typical CRISPR diagnostics require a nucleic acid pre-amplification step prior to assay readout, this thesis presents optimized CRISPR/Cas reaction conditions and nanozyme functionalization strategies to enable the detection of non-coding RNAs without a nucleic acid pre-amplification step. The developed CrisprZyme was clinically evaluated by showcasing the ability to distinguish healthy from unhealthy patients that arrived in hospital emergency rooms with chest pain and detecting a biomarker from tumor biopsies from patients with prostate cancer. (ii) As an alternative readout mechanism, a polymerization-amplified CRISPR assay for nucleic acid detection (Pacman) was also explored. This second platform consisted of a hemin-loaded DNA flower (DNF) that was enzymatically cleaved by a CRISPR-activated Cas12a. The process liberated hemin from the network into a solution that contained reagents for polymerization. Hemin acted as the catalyst for the reaction allowing a visual readout based on turbidity for PoC diagnostics. The assay was employed for the detection of SARS-CoV-2 and ZIKA virus by the naked eye, with a detection limit down to 5 copies/µL when coupled with a pre-amplification step...
Version
Open Access
Date Issued
2023-03-12
Date Awarded
01/09/2023
License URL
Advisor
Stevens, Molly
Rifaie-Graham, Omar
Najer, Adrian
Sponsor
Singapore. Agency for Science, Technology and Research
Grant Number
NSS PhD
Publisher Department
Materials
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)