Nucleic acid-based theranostics

• Main Author: Liu, Mengying
• Other Authors: Tan Meng How
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/171349
• Institution: Nanyang Technological University

The outbreak of COVID-19 pandemic has caused over seven hundred million positive cases and over seven million deaths worldwide as reported on the WHO website (https://covid19.who.int/). Due to the rapid transmission and highly pathogenicity, it has posed tremendously negative effects on people’s life as well as hampered social and economy development. A variety of new technologies have been developed during this COVID-19 pandemic era for COVID-19 diagnostics as well as treatment. However, most of them have a long and complicated workflow and rely on sophisticated equipment and trained personnel, thus they are not practical to be applied to remote and poor communities for COVID-19 detection. While Antigen Rapid test (ART) serves as a regular point-of-care test (POCT) , it suffers from poor sensitivity. Although Isothermal amplification-based methodologies have a relatively high sensitivity and it can be conducted in single temperature, they prone to give false positive results because there are some spurious by-products generated in the reactions. To address all the issues mentioned above, by working with other lab members and collaborators , three novel nuclei based diagnostic assays (named VaNGuard, LANTERN and SNiPRE ) for COVID-19 detection and one CRISPR-CasRx based RNA base editor (xPERT) which holds promising potential for preventing COVID-19 infection and repairing other disease relevant mutations were developed. All three COVID-19 diagnostic assays have been clinically validated . They all have a high sensitivity and are able to give accurate test results for crude samples without RNA isolation steps within 30 minutes. They all can be used for POCT because they can provide accurate test results either via dipsticks version or on a potable home-made equipment. Additionally, they all have their own specialized merits. CRISPR-based VaNGuard assay is able to detect SARS-CoV-2 even there were some certain mutations on its targets. LANTERN assay is a probe-based assay and has a Limitation of Detection (LoD ) of 8 copies per reaction. It also enables robust one-pot reaction happening even when a human internal control was incorporated. As a variety of COVID-19 variants started to emerge, our three-probe version of SNiPRE assay enables rapid and accurate COVID-19 variants detection with a LoD of 20 copies per reaction and a comprehensive output for the interpretation of individual infection statues. Many RNA base editors have suffered either a poor performance for on target editing or producing many unwanted and non-specific edits. By systematic optimization on both deaminase domain and Cas scaffold, xPERT was engineered into a competent RNA base editor which has both high on-target editing efficiency as well as low off-target activities. In addition, unlike other proposed nuclei acid based therapeutic approaches such as siRNA or wild-type Cas enzymes , which merely reply on virus degradation for COVID-19 treatment, our xPERT construct exhibited the potential therapeutic value through modifying the ACE2 receptor, which is the key entry for SARS-CoV-2 to enter human cells. Our xPERT work had demonstrated the promising capacity of xPERT and fusion gRNAs for blocking SARS-CoV-2 infection without affecting other important biological function of ACE2 receptors.