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

• DOI: 10.1038/s41467-021-21996-6
• Publication Date: 2021-03-19T13:05:27Z
• Authors: Kean Hean Ooi; Mengying Mandy Liu; Jie Wen Douglas Tay; Seok Yee Teo; Pornchai Kaewsapsak; Shengyang Jin; Chun Kiat Lee; Jingwen Hou; Sebastian Maurer-...; Weisi Lin; Benedict Yan; Gabriel Yan; Yong-Gui Gao; Meng How Tan
• Keywords: 0301 basic medicine; Endodeoxyribonucleases; SARS-CoV-2; Science; Q; CRISPR-Associated Proteins; COVID-19; Sensitivity and Specificity; Article; 3. Good health; 03 medical and health sciences; COVID-19 Testing; Bacterial Proteins; Molecular Diagnostic Techniques; Nasopharynx; Mutation; Humans; RNA, Viral; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; Nucleic Acid Amplification Techniques; RNA, Guide, Kinetoplastida

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