Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis

Abstract

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

Keywords: COVID-19; CRISPR; Fluorescent detection; Highly sensitive; Molecular diagnosis; SARS-CoV-2.

Fig. 1

A CRISPR-based Fluorescent Diagnosis System for COVID-19 (COVID-19 CRISPR-FDS). (A) Schematic illustration of a CRISPR-FDS assay for detection of SARS-CoV-2 RNA in clinical samples. (B) SARS-CoV-2 genome map of COVID-19 CRISPR-FDS target sequences, and (C) sites in ORF1ab gene and the N protein gene that are detected COVID-19 CRISPR-FDS. Normalized CRISPR-FDS photoluminescent (PL) signal from SARS-CoV-2 RNA positive (10⁹ copies/sample) and negative control (polyA carrier RNA) samples following (D) target amplification by RT-PCR or RPA, (E) by RT-PCR for each assay target, and (F) by RT-PCR for related beta coronavirus species (10⁹ copies/sample). Bar graph data represents the mean ± SD, of three experimental replicates.

Fig. 2

COVID-19 CRISPR-FDS assay optimization. (A) Substrate-dependent, (B) temperature-dependent, and (C) target-dependent effects on CRISPR-FDS signal. An aliquot containing 10⁹ target amplicon copies, or an equivalent amount of poly A carrier RNA were analyzed as positive (+) and negative (−) control samples, respectively. Data presented in the top rows of each panel and the bottom row of (C) are normalized to the highest signal intensity detected in the corresponding experiment. Bar graph data represents the mean ± SD, of three experimental replicates. (ns, P > 0.05; ****,P < 0.0001).

Fig. 3

COVID-19 CRISPR-FDS analytical and diagnostic performance. Limit of detection (LOD) samples containing the indicated number of viral genomes after amplification by (A) RT-PCR and (B) RT-RPA for COVID-19 CRISPR-FDS analysis or by (C) RT-qPCR, indicated significant differences and undetermined (UD) results. (D) RT-PCR COVID-19 CRISPR-FDS results for a cohort of 29 individuals with suspected COVID-19 cases, run in parallel with blank (BC; nuclease free water), negative (NC; carrier RNA) and positive (PC; 10⁹ target amplicon copies) control samples, where the dashed line indicates the threshold for a positive result. Results depict the mean ± SD of three experimental replicates. (E) Comparison of SARS-CoV-2 test results for matching patient samples analyzed by CRISPR-FDS, or by RT-qPCR by a state (qPCR 1) and a clinical testing laboratory (qPCR 2). (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****,P < 0.0001).