Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities
Zhijian Yi,
Jean de Dieu Habimana,
Omar Mukama,
Zhiyuan Li,
Nelson Odiwuor,
Hanzhi Jing,
Chengrong Nie,
Mei Hu,
Zuoxian Lin,
Hongping Wei,
Lingwen Zeng
Affiliations
Zhijian Yi
School of Food Science and Engineering, Foshan University, Foshan 528231, China
Jean de Dieu Habimana
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Omar Mukama
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Zhiyuan Li
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Nelson Odiwuor
University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049, China
Hanzhi Jing
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Chengrong Nie
School of Food Science and Engineering, Foshan University, Foshan 528231, China
Mei Hu
College of Food Science and Technology, Henan Agricultural University, 63 Nongye Road, Zhengzhou 450002, China
Zuoxian Lin
Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Hongping Wei
University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049, China
Lingwen Zeng
School of Food Science and Engineering, Foshan University, Foshan 528231, China
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.
