CRISPR-Cas13a Cascade-based Viral RNA Assay for Detecting SARS-CoV-2 and Its Mutations in Clinical Samples

Overview

Journal Sens Actuators B Chem

Date 2022 Apr 4

PMID 35370361

Authors

Yuxi Wang

Ting Xue

Minjin Wang

Rodrigo Ledesma-Amaro

Ying Lu

Xinyue Hu

Ting Zhang

Ming Yang

Yalun Li

Jin Xiang

Ruijie Deng

Binwu Ying

Weimin Li

Affiliations

Soon will be listed here.

Abstract

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

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