An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
Kean Hean Ooi,
Mengying Mandy Liu,
Jie Wen Douglas Tay,
Seok Yee Teo,
Pornchai Kaewsapsak,
Shengyang Jin,
Chun Kiat Lee,
Jingwen Hou,
Sebastian Maurer-Stroh,
Weisi Lin,
Benedict Yan,
Gabriel Yan,
Yong-Gui Gao and
Meng How Tan ()
Additional contact information
Kean Hean Ooi: Nanyang Technological University
Mengying Mandy Liu: Nanyang Technological University
Jie Wen Douglas Tay: Nanyang Technological University
Seok Yee Teo: Nanyang Technological University
Pornchai Kaewsapsak: Genome Institute of Singapore, Agency for Science Technology and Research
Shengyang Jin: Nanyang Technological University
Chun Kiat Lee: National University Health System
Jingwen Hou: Nanyang Technological University
Sebastian Maurer-Stroh: Bioinformatics Institute, Agency for Science Technology and Research
Weisi Lin: Nanyang Technological University
Benedict Yan: National University Health System
Gabriel Yan: National University Hospital, National University Health System
Yong-Gui Gao: Nanyang Technological University
Meng How Tan: Nanyang Technological University
Nature Communications, 2021, vol. 12, issue 1, 1-23
Abstract:
Abstract 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.
Date: 2021
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-21996-6
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DOI: 10.1038/s41467-021-21996-6
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