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Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2
Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we i...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9282225/ https://www.ncbi.nlm.nih.gov/pubmed/35763567 http://dx.doi.org/10.1073/pnas.2118260119 |
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author | Mahas, Ahmed Marsic, Tin Lopez-Portillo Masson, Mauricio Wang, Qiaochu Aman, Rashid Zheng, Cheng Ali, Zahir Alsanea, Madain Al-Qahtani, Ahmed Ghanem, Bernard Alhamlan, Fatimah Mahfouz, Magdy |
author_facet | Mahas, Ahmed Marsic, Tin Lopez-Portillo Masson, Mauricio Wang, Qiaochu Aman, Rashid Zheng, Cheng Ali, Zahir Alsanea, Madain Al-Qahtani, Ahmed Ghanem, Bernard Alhamlan, Fatimah Mahfouz, Magdy |
author_sort | Mahas, Ahmed |
collection | PubMed |
description | Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies. |
format | Online Article Text |
id | pubmed-9282225 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-92822252022-07-15 Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 Mahas, Ahmed Marsic, Tin Lopez-Portillo Masson, Mauricio Wang, Qiaochu Aman, Rashid Zheng, Cheng Ali, Zahir Alsanea, Madain Al-Qahtani, Ahmed Ghanem, Bernard Alhamlan, Fatimah Mahfouz, Magdy Proc Natl Acad Sci U S A Biological Sciences Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/μL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies. National Academy of Sciences 2022-06-28 2022-07-12 /pmc/articles/PMC9282225/ /pubmed/35763567 http://dx.doi.org/10.1073/pnas.2118260119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Biological Sciences Mahas, Ahmed Marsic, Tin Lopez-Portillo Masson, Mauricio Wang, Qiaochu Aman, Rashid Zheng, Cheng Ali, Zahir Alsanea, Madain Al-Qahtani, Ahmed Ghanem, Bernard Alhamlan, Fatimah Mahfouz, Magdy Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title | Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title_full | Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title_fullStr | Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title_full_unstemmed | Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title_short | Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2 |
title_sort | characterization of a thermostable cas13 enzyme for one-pot detection of sars-cov-2 |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9282225/ https://www.ncbi.nlm.nih.gov/pubmed/35763567 http://dx.doi.org/10.1073/pnas.2118260119 |
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