Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2

Tin Marsic; Zahir Ali; Muhammad Tehseen; Ahmed Mahas; Samir Hamdan; Magdy M. Mahfouz

doi:10.1021/acs.nanolett.1c00612

Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2

Tin Marsic, Zahir Ali, Muhammad Tehseen, Ahmed Mahas, Samir Hamdan, Magdy M. Mahfouz

Research output: Contribution to journal › Article › peer-review

59 Scopus citations

Abstract

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

Original language	English (US)
Journal	Nano Letters
DOIs	https://doi.org/10.1021/acs.nanolett.1c00612
State	Published - Apr 12 2021

Bibliographical note

KAUST Repository Item: Exported on 2021-04-19
Acknowledgements: We would like to thank Mohammad Alarawi for providing the RNA of SARS-CoV-2 clinical samples. We also thank members of the genome engineering and synthetic biology laboratory for insightful discussions and technical support. This work was supported, in part, by the Smart Health Initiative at KAUST and the IAF grant from the KAUST IED to M.M.

ASJC Scopus subject areas

Bioengineering
General Materials Science
General Chemistry
Mechanical Engineering
Condensed Matter Physics

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10.1021/acs.nanolett.1c00612

https://repository.kaust.edu.sa/bitstream/10754/668815/1/acs.nanolett.1c00612.pdf

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title = "Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2",

abstract = "Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.",

author = "Tin Marsic and Zahir Ali and Muhammad Tehseen and Ahmed Mahas and Samir Hamdan and Mahfouz, {Magdy M.}",

note = "KAUST Repository Item: Exported on 2021-04-19 Acknowledgements: We would like to thank Mohammad Alarawi for providing the RNA of SARS-CoV-2 clinical samples. We also thank members of the genome engineering and synthetic biology laboratory for insightful discussions and technical support. This work was supported, in part, by the Smart Health Initiative at KAUST and the IAF grant from the KAUST IED to M.M.",

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language = "English (US)",

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AU - Marsic, Tin

AU - Ali, Zahir

AU - Tehseen, Muhammad

AU - Mahas, Ahmed

AU - Hamdan, Samir

AU - Mahfouz, Magdy M.

N1 - KAUST Repository Item: Exported on 2021-04-19 Acknowledgements: We would like to thank Mohammad Alarawi for providing the RNA of SARS-CoV-2 clinical samples. We also thank members of the genome engineering and synthetic biology laboratory for insightful discussions and technical support. This work was supported, in part, by the Smart Health Initiative at KAUST and the IAF grant from the KAUST IED to M.M.

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N2 - Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

AB - Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

UR - http://hdl.handle.net/10754/668815

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JF - Nano Letters

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Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2

Abstract

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