Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Abstract

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

Nanotechnology-based diagnostic methods for coronavirus: From nucleic acid extraction to amplification

“…Based on this detection mechanism, the whole assay took ∼45 min and the limit of detection is 10 copies per μL. Furthermore, Yi et al designed a DNA capture probe-based LFA strip for the detection of SARS-CoV-2 ( Yi et al, 2021 ). Compared to conventional LFA strips, streptavidin-modified AuNPs instead of anti-FAM antibody-AuNPs acted as signal probes for the naked-eye readout.…”

REASSURED Multiplex Diagnostics: A Critical Review and Forecast

“…It has the potential for multiplexing and is able to produce results in about an hour. Yi et al presented a similar system termed CRICOLAP for the detection of SARS-CoV-2 and also employs an amplification step by RT-LAMP, which is followed by a CRISPR/Cas12a collateral cleavage system for target recognition [ 107 ]. The paper reports a real-time parallel fluorescent readout system.…”

A loop-mediated isothermal amplification-enabled analytical assay for the detection of SARS-CoV-2: A review

“…Compared with qRT-PCR, this method has reliable accuracy and was verified to be feasible (Broughton et al, 2020). A novel DNA capture probe-based LFA for detecting SARS-CoV-2 was proposed (Yi et al, 2021). First, based on a CRISPR/Cas12a detection method, a biotinylated reporter gene was designed according to the S and N genes of SARS-CoV-2.…”