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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
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.
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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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