Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2

Abstract

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Keywords: COVID-19; CRISPR; SARS-CoV-2; diagnosis; fluorescence detection; lysis buffer.

Figure 1

Lyo-CRISPR SARS-CoV-2 assay. (A) Processing scheme, including the reagents and equipment required for performing the following three steps: RNA extraction using spin columns or by treating the samples with lysis buffer and heat, to be used as input in the reverse transcription (RT) and isothermal amplification reactions and CRISPR-Cas12 detection for N gene by fluorescence. Resuspension with nuclease-free water is required to hydrate the lyophilized beads containing all components for isothermal amplification and CRISPR detection. (B) Workflow scheme showing the entire process at molecular level. PBS: Phosphate Buffered Saline buffer; min: minutes; Lyo: lyophilized; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; CRISPR: clustered regularly interspaced short palindromic repeats; sgRNA: single guide RNA; F-Q reporter: fluorophore-quencher reporter.

Figure 2

Lyo-CRISPR SARS-CoV-2 kit results of 210 RNA samples extracted from nasopharyngeal swabs (NPS) using spin columns (A–C) and 30 RNA samples extracted from nasopharyngeal swabs with direct lysis (D–F). (A,D) Lyo-CRISPR SARS-CoV-2 kit results for N gene and internal control (RNAseP) in RT-qPCR-negative (grey) and -positive (blue) samples. Median, interquartile range, and range of the fluorescence ratio (R = intensity fluorescence (IF)_t20 sample/IF_t20 non-template control) values measured at 20 min are shown. A positive result was considered if R ≥ 2.5 or negative if R ˂ 2.5. (B,E) R values obtained for each RT-qPCR-negative (grey) and -positive (blue) sample tested. Each dot represents one sample and the dashed line represents the cutoff of the Lyo-CRISPR test to classify them. Positive samples were sorted by Ct values determined using the GeneFinder RT-qPCR kit (N gene). (C,F) R values obtained for each RT-qPCR-positive sample tested. Each dot represents one sample. N: nucleocapsid; Id: identification; Ct: Cycle threshold, R: fluorescence ratio. Figures were designed with GraphPad Prism V8 software (GraphPad, San Diego, California, USA) and BioRender.com.