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

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) specime...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979722/
https://www.ncbi.nlm.nih.gov/pubmed/33741959
http://dx.doi.org/10.1038/s41467-021-21996-6
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author 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
author_facet 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
author_sort Ooi, Kean Hean
collection PubMed
description 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.
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spelling pubmed-79797222021-04-16 An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing 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 Nat Commun Article 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. Nature Publishing Group UK 2021-03-19 /pmc/articles/PMC7979722/ /pubmed/33741959 http://dx.doi.org/10.1038/s41467-021-21996-6 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
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
An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title_full An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title_fullStr An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title_full_unstemmed An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title_short An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
title_sort engineered crispr-cas12a variant and dna-rna hybrid guides enable robust and rapid covid-19 testing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979722/
https://www.ncbi.nlm.nih.gov/pubmed/33741959
http://dx.doi.org/10.1038/s41467-021-21996-6
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