An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

• Published in: Nature communications 2021-03, Vol.12 (1), p.1739-23, Article 1739
• Main Authors: Ooi, Kean Hean, Liu, Mengying Mandy, Tay, Jie Wen Douglas, Teo, Seok Yee, Kaewsapsak, Pornchai, Jin, Shengyang, Lee, Chun Kiat, Hou, Jingwen, Maurer-Stroh, Sebastian, Lin, Weisi, Yan, Benedict, Yan, Gabriel, Gao, Yong-Gui, Tan, Meng How
• Format: Article
• Language: English
• Subjects: 14/10; 38/88; 42/34; 45/71; 49/90; 631/1647/2196/2197; 631/326/596/4130; 631/337/4041/3196; Bacterial Proteins - genetics; Clustered Regularly Interspaced Short Palindromic Repeats; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 - virology; COVID-19 diagnostic tests; COVID-19 Testing - methods; CRISPR; CRISPR-Associated Proteins - genetics; CRISPR-Cas Systems; Deoxyribonucleic acid; Diagnostic systems; Disease transmission; DNA; Endodeoxyribonucleases - genetics; Gene amplification; Genomes; Humanities and Social Sciences; Humans; Molecular Diagnostic Techniques - methods; multidisciplinary; Mutation; Nasopharynx - virology; Nucleic Acid Amplification Techniques - methods; Pandemics; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; RNA, Viral - genetics; Robustness; SARS-CoV-2 - genetics; Science; Science (multidisciplinary); Sensitivity and Specificity; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Viral diseases; Viruses
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-21996-6

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively. As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.