Developing a rapid, specific and sensitive CRISPR/Cas12a-based test kit for in-vitro diagnosis of COVID-19

• Main Author: Teo, Seok Yee
• Other Authors: Gao Yonggui
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences::Molecular biology; Science::Biological sciences::Genetics
• Online Access: https://hdl.handle.net/10356/140518
• Institution: Nanyang Technological University

SARS-CoV-2 is the causative agent of the ongoing COVID-19 pandemic. The outbreak originated in Wuhan, China, and has since spread throughout the world. Given the severity of disease and high transmission potential of SARS-CoV-2, rapid, specific and sensitive assays are required to meet diagnostic needs related to the outbreak. A recent report has described the use of a Cas12a-based method to detect SARS-CoV-2 with high specificity and sensitivity. However, the workflow is time-consuming and require multiple heating sources, making it incompatible for field application. Here, we improve the field usability of Cas12a detection by using a fluorescence-based screening assay to identify a Cas12a protein, enAsCas12a, that can specifically detect SARS-CoV-2 at room temperature. Using mismatched gRNA, we found that enAsCas12a can tolerate single-nucleotide mismatches throughout the target sequence, making it robust against viral evolution. In addition, RT-LAMP-Cas12a detection conditions were optimized to reduce overall sample-to-result time by 8 min without compromising sensitivity. Although results are inconclusive, we demonstrate that Cas12a detection may be visualized on a lateral flow strip for easy result interpretation. Finally, different gRNA targeting SARS-CoV-2 were screened that show higher signals than gRNA previously reported for SARS-CoV-2 detection.