iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

Keywords: Biosensors; COVID-19; CRISPR-Cas12; Diagnostics; Nucleic acid detection; RT-LAMP; SARS-CoV-2.

Fig. 1

CRISPR–Cas12a-based iSCAN assay for detection of SARS-CoV-2. A-A schematic view of the SARS-CoV-2 genome architecture. Regions targeted by iSCAN assay are highlighted. B-Workflow of iSCAN detection assay. C-Quantifications of signal intensities of CRISPR-Cas12a fluorescence-based detection assays of synthetic SARS-CoV-2. E-T: E gene with Tris buffer, E-H: E gene with HEPES buffer, CE: E gene with commercial enzymes (NEB), N1: no crRNA, N2: ns-crRNA: nonspecific cRNA, NTC: no-template control. D-Limit of detection (LoD) determination of RT-LAMP-Cas12a assay. Values shown as mean ± SEM (n = 4) E-End-point fluorescence visualization under UV light following the CRISPR/Cas12a detection assay performed on clinical samples. S: different clinical samples with different Ct values, +ve: Synthetic RNA, NTC: no-template control. F-Lateral flow readouts of Cas12a detection of SARS-CoV-2 RNA in clinical samples G-End-point fluorescence intensity measured in clinical samples after CRISPR/Cas12a detection assay.

Fig. 2

CRISPR–Cas12b-based assay for detection of SARS-CoV-2. A-Limit of detection (LoD) of RT-LAMP-AapCas12b assay in spotted one-pot reaction. Values shown as mean ± SEM (n = 3). B-Visual fluorescence output of four clinical samples under UV light following CRISPR/Cas12b detection assay performed by two different strategies. S: different clinical samples with different Ct values. C-End-point fluorescence intensity measured in clinical samples after performing spotted one-pot CRISPR-AapCas12a detection assay. S: different clinical samples with different Ct values, +Ve: Synthetic RNA, NTC: no-template control. D-Lateral flow readout after spotted one-pot AapCas12b detection assay performed on clinical samples.

Fig. 3

Overview of the CRISPR-Cas based SARS-CoV-2 detection methods and requirements. A) Schematic illustration showing different Cas12-based detection modalities. The first row depicts Cas12a-based two-pot reaction, where the RT-LAMP amplification of SARS-CoV-2 nucleic acid is performed in the first tube. Then the RT-LAMP product is transferred to a second tube for Cas12a-based detection. The second row depicts RT-LAMP and Cas12b-based detection reagents, except for Cas12b-sgRNA complex, mixed in a single reaction. Cas12b-sgRNA complex is temporary separated from the reaction because it is added on the tube wall. After 30 min of RT-LAMP reaction, the Cas12b-sgRNA complex is centrifuged into the reaction mix for target cleavage and CRISPR/Cas-based detection, which takes place for an additional 15 min. In the third row, simultaneous RT-LAMP amplification and CRISPR-based detection of SARS-CoV-2 detection is performed in a single tube. B) Minimal equipment and consumables required for iSCAN detection assay, which includes a heat block, pipettes, pipette tips, sample tubes, and lateral flow strips.