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.

Citation
Marsic, T., Ali, Z., Tehseen, M., Mahas, A., Hamdan, S., & Mahfouz, M. (2021). Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2. Nano Letters. doi:10.1021/acs.nanolett.1c00612

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.

Publisher
American Chemical Society (ACS)

Journal
Nano Letters

DOI
10.1021/acs.nanolett.1c00612

PubMed ID
33844549

Additional Links
https://pubs.acs.org/doi/10.1021/acs.nanolett.1c00612

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