Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19

Abstract

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.

Fig 1

(A) Number of mNGS reads mapped to the genome of SARS-CoV-2 in 52 positive cases; (B) Phylogenetic tree of SARS-CoV-2s and other pathogenic CoVs; (C) SARS-CoV-2 genome and CRISPR target region. Nucleotide identity among the SARS-CoV-2 genomes, including 52 in our cohort and 2,439 from public databases (Blue), between SARS-CoV-2 (NC_045512.2) and other pathogenic CoVs (Red, SARS-CoV; others as indicated). Brief gene locations are presented above and the target region for CRISPR-COVID is indicated in Grey.

Fig 2. Analytical assessment of the sensitivity and specificity of CRISPR-COVID.

(A) Schematic diagram of CRISPR-COVID. The collateral nuclease activity of Cas proteins is activated upon specific binding of gRNA to the Orf1ab gene. Fluorescent signal produced from cleaved probes is captured and indicates the presence of SARS-CoV-2; Analytic evaluation of assay performance by testing contrived negative swab samples with indicated titers of SARS-CoV-2 (B), and various microbes as interfering materials. (CRBP, Common respiratory bacterial pathogens; CRVP, Common respiratory viral pathogens; 1, S. pneumoniae; 2, H. influenzae; 3, M. pneumoniae; 4, C. pneumoniae; 5, B. pertusiss, 6, S. mitis; 7, S. pyogenes; 8, S. aureus; 9, E. coli; 10, E. faecalis; 11, hCoV-OC43; 12, hCoV-NL63; 13, hCoV-HKU-1; 14, hCoV-229E; 15, Adenovirus Type 3; 16, H. influenzae B (Victoria); 17, H. influenzae A (H3N2); 18, HPIV-1; 19, RSV-A) (C). Group Blue, common respiratory bacterial pathogens; Green, Other bacteria; Brown, other CoVs; Yellow, common respiratory viral pathogens. P < 0.001 by Student's t-test between PC and all other samples.

Fig 3. Summary of the cohort and CRISPR-COVID results.

(A) A total of 112 specimens included in this study, including 52 cases positive for SARS-CoV-2 and 62 negative cases. (B) Results of CRISPR-COVID in different sample groups. Positives and negatives were called based on the fold change and cutoff values; (C) Result summary on the suspected SARS-CoV-2 samples by mNGS, CRISPR-COVID and PCR-COVID. P = 0.056 between mNGS and RT-PCR, and between CRISPR and RT-PCR by Fisher's exact test.

Fig 4. Kaplan–Meier curve of CRISPR-COVID positive rate by CRISPR and PCR.

* Log-rank test, P < 0.05.