RAY: CRISPR diagnostic for rapid and accurate detection of SARS-CoV2 variants on a paper strip

Abstract

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations- some of which have recently been linked with increased viral load and prognostic complexities. Interestingly, several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qPCR) method. This necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. In this study, we show that the high specificity of Francisella novicida Cas9 (FnCas9) to point mismatches can be successfully adapted for the simultaneous detection of SARS-CoV2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the mutation N501Y (earlier shown to be present in the British N501Y.V1, South African N501Y.V2, and Brazilian N501Y.V3 variants of SARS-CoV2) within an hour using paper strip chemistry. The results were corroborated using deep sequencing. Our design principle can be rapidly adapted for other mutations, highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19.

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

“…CRISPR-based variant detection techniques focus on designing crRNAs with mismatches for mutation positions in known sequences 498,499 or developing chimeric crRNAs to distinguish mutations in different positions specically. 500 Most of these technologies use uorescence 501 or LFAs 502 for the readout of the detection results, and some have integrated technology into micro devices to develop POCT devices that integrate extraction, purication, and concentration of viral RNA, amplication and detection. 503 CRISPR technology has the merits of easy-to-use and easy-to-set-up, which can be rapidly deployed in response to outbreaks.…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…Specificity: Unclear [309] Almost all variants 69del, 70del, Y144del, N501Y and A570D Compared to WGS, RT-PCR and sanger sequencing require shorter time and lower cost. LOD unclear [310] SNP genotyping Almost all variants Unreported >62% to be able to distinguish two variants of different genotypes [311] RT-LAMP P.1 (Gamma), P.2 (Zeta) N501Y, E484K/Q, K417N/T Sensitivity: 97%, Specificity: 100% (based on N/E targets) [312] B.1.1.7 (Alpha) 69del, 70del LOD: 39-10,000 RNA copies/reaction [313] CRISRP/Cas-based technique B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) N501Y LOD, sensitivity and specificity unclear [346] Almost all variants have D614G D614G LOD: 10 RNA copies/reaction [314] Other variants E174R/S542R/K548R, S254F LOD: 50-1000 RNA copies/reaction, specificity: 100% [315] Almost all variants have D614G D614G LOD: 82 RNA copies/reaction, % specificity unclear [155] Antigen test B.1.1.7 (Alpha), B.1.351 (Beta), Unclear LOD: 1.7×10 5 – 6.6×10 7 RNA copies/mL [316] Sensitivity and specificity: Unclear Antigen and antibody test B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) Unclear Detection rate of antigen/antibody tests to detect these three variants were reported to be >90% [317] …”