Evaluation of Cas12a-gRNA pairs for the rapid detection of SARS-CoV-2 with CRISPR diagnostics

• Main Author: Tay, Douglas Jie Wen
• Other Authors: Tan Meng How
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/140677
• Institution: Nanyang Technological University

The ongoing COVID-19 pandemic is caused by SARS-CoV-2 of zoonotic origin. As of 1 May 2020, there were 3,175,207 confirmed cases and 224,172 deaths globally, raising great public concerns worldwide. A cornerstone of managing the COVID-19 pandemic is through diagnostic testing to detect community clusters and interrupt disease transmission. The current reference standard is based on qRT-PCR, which are limited by the requirement for thermocycling conditions (hence specialised equipment), long turnaround time (~90 min excluding nucleic acid extraction) and trained operators. The collateral activity of CRISPR-Cas has recently been harnessed for the development of a rapid (<30 min), specific and sensitive nucleic acid detection platform. We designed gRNA targets to the ORF1ab and S genes of SARS-CoV-2, followed by detection with five Cas12a variants using a ssDNA fluorophore-quencher reporter to determine the best Cas12a-gRNA pair. Our results indicate enAsCas12a:S-2 as the best candidate for follow-up, with high fluorescence signal and mismatch tolerance for the detection of variable SARS-CoV-2 genomes. No cross-reactivity was observed with related coronaviruses (SARS-CoV and MERS-CoV), suggesting specificity for SARS-CoV-2.