CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples

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

doi:10.1016/j.snb.2022.131765

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples

Sens Actuators B Chem. 2022 Jul 1:362:131765. doi: 10.1016/j.snb.2022.131765. Epub 2022 Mar 27.

Authors

Yuxi Wang^{1

2}, Ting Xue¹, Minjin Wang³, Rodrigo Ledesma-Amaro⁴, Ying Lu^{5

6}, Xinyue Hu^{2

6}, Ting Zhang⁵, Ming Yang¹, Yalun Li¹, Jin Xiang², Ruijie Deng⁵, Binwu Ying³, Weimin Li^{1

2}

Affiliations

¹ Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu 610041, China.
² Targeted Tracer Research and development laboratory, Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
³ Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu 610041, China.
⁴ Department of Bioengineering, Imperial College Centre for Synthetic Biology, Imperial College London, London, UK.
⁵ College of Biomass Science and Engineering, Sichuan University, Chengdu 610065, China.
⁶ State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.

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.

Keywords: COVID-19; COVID-19, coronavirus disease 2019; CRISPR, clustered regularly interspaced short palindromic repeats; CRISPR-Cas13; Cas, CRISPR associated proteins; LOD, limit of detection; NASBA, nucleic acid sequence-based amplification; Nucleic acid tests; RNA aptamer; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; T4 PNK, T4 Polynucleotide Kinase; crRNA, CRISPR RNA.