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

## 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.

## Key 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 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.