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Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediate...

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Autores principales: Santiago-Frangos, Andrew, Hall, Laina N., Nemudraia, Anna, Nemudryi, Artem, Krishna, Pushya, Wiegand, Tanner, Wilkinson, Royce A., Snyder, Deann T., Hedges, Jodi F., Cicha, Calvin, Lee, Helen H., Graham, Ava, Jutila, Mark A., Taylor, Matthew P., Wiedenheft, Blake
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157118/
https://www.ncbi.nlm.nih.gov/pubmed/34075364
http://dx.doi.org/10.1016/j.xcrm.2021.100319
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author Santiago-Frangos, Andrew
Hall, Laina N.
Nemudraia, Anna
Nemudryi, Artem
Krishna, Pushya
Wiegand, Tanner
Wilkinson, Royce A.
Snyder, Deann T.
Hedges, Jodi F.
Cicha, Calvin
Lee, Helen H.
Graham, Ava
Jutila, Mark A.
Taylor, Matthew P.
Wiedenheft, Blake
author_facet Santiago-Frangos, Andrew
Hall, Laina N.
Nemudraia, Anna
Nemudryi, Artem
Krishna, Pushya
Wiegand, Tanner
Wilkinson, Royce A.
Snyder, Deann T.
Hedges, Jodi F.
Cicha, Calvin
Lee, Helen H.
Graham, Ava
Jutila, Mark A.
Taylor, Matthew P.
Wiedenheft, Blake
author_sort Santiago-Frangos, Andrew
collection PubMed
description There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 10(7) copies/μL for a single guide RNA to 10(6) copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.
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spelling pubmed-81571182021-05-28 Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic Santiago-Frangos, Andrew Hall, Laina N. Nemudraia, Anna Nemudryi, Artem Krishna, Pushya Wiegand, Tanner Wilkinson, Royce A. Snyder, Deann T. Hedges, Jodi F. Cicha, Calvin Lee, Helen H. Graham, Ava Jutila, Mark A. Taylor, Matthew P. Wiedenheft, Blake Cell Rep Med Report There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 10(7) copies/μL for a single guide RNA to 10(6) copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min. Elsevier 2021-05-27 /pmc/articles/PMC8157118/ /pubmed/34075364 http://dx.doi.org/10.1016/j.xcrm.2021.100319 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Report
Santiago-Frangos, Andrew
Hall, Laina N.
Nemudraia, Anna
Nemudryi, Artem
Krishna, Pushya
Wiegand, Tanner
Wilkinson, Royce A.
Snyder, Deann T.
Hedges, Jodi F.
Cicha, Calvin
Lee, Helen H.
Graham, Ava
Jutila, Mark A.
Taylor, Matthew P.
Wiedenheft, Blake
Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title_full Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title_fullStr Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title_full_unstemmed Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title_short Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
title_sort intrinsic signal amplification by type iii crispr-cas systems provides a sequence-specific sars-cov-2 diagnostic
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157118/
https://www.ncbi.nlm.nih.gov/pubmed/34075364
http://dx.doi.org/10.1016/j.xcrm.2021.100319
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