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 Mahfouz
TL;DR
This paper introduces Vigilant, a new method combining CRISPR and lateral flow assays to detect SARS-CoV2 with high sensitivity and specificity.
Contribution
The novel fusion of VirD2 relaxase and dCas9 enables sensitive and specific nucleic acid detection via lateral flow assays.
Findings
Vigilant detects SARS-CoV2 with a limit of detection of 2.5 copies/μL.
The method shows no cross-reactivity with SARS-CoV1 or MERS.
Vigilant is a rapid, cost-effective, and robust platform for point-of-care testing.
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…
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Taxonomy
TopicsCRISPR and Genetic Engineering · Advanced biosensing and bioanalysis techniques · SARS-CoV-2 detection and testing