FnCas9 based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV2 variants on a paper strip
- CSIR Institute of Genomics and Integrative Biology, India
- CSIR-Institute of Genomics and Integrative Biology, India
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. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that 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 S gene mutation N501Y (present across multiple variant lineages of SARS-CoV2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild type (n=37) and mutant (n=22) viral RNA samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.
Data availability
Sequencing data associated with the manuscript have been deposited to GISAID with the following numbers:EPI_ISL_911542, EPI_ISL_911532, EPI_ISL_911543, EPI_ISL_911533, EPI_ISL_911544, EPI_ISL_911534, EPI_ISL_911545, EPI_ISL_911535, EPI_ISL_911546, EPI_ISL_911536, EPI_ISL_911547, EPI_ISL_911537, EPI_ISL_911538, EPI_ISL_911540, EPI_ISL_911541, EPI_ISL_911539 were just released and are now available to all participants in GISAID.
Article and author information
Author details
Janani Srinivasa Vasudevan
Debojyoti Chakraborty
Funding
University Grants Commission (Graduate student fellowship)
- Manoj Kumar
IUSSTF (CLP-0033)
- Rajesh Pandey
CSIR Sickle Cell Anemia Mission (HCP0008)
- Debojyoti Chakraborty
Tata Steel (SSP 2001)
- Debojyoti Chakraborty
Lady Tata Young Investigator (GAP0198)
- Debojyoti Chakraborty
CSIR Sickle Cell Anemia Mission (HCP0008)
- Souvik Maiti
CSIR (Graduate Student fellowship)
- Mohd Azhar
CSIR (Graduate Student fellowship)
- Jayaram Murthy
CSIR (Research Associateship)
- Sneha Gulati
Indian Council of Medical Research (Graduate Student fellowship)
- Asgar H Ansari
CSIR (Graduate Student fellowship)
- Rhythm Phutela
CSIR (Graduate Student fellowship)
- Sundaram Acharya
CSIR (Research Associateship)
- Poorti Kathpalia
CSIR (MLP 2005)
- Rajesh Pandey
Fondation Botnar (CLP-0031)
- Rajesh Pandey
Intel Corporation (CLP-0034)
- Rajesh Pandey
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Yamuna Krishnan, University of Chicago, United States
Ethics
Human subjects: The present study was approved by the Ethics Committee, Institute of Genomics and Integrative Biology, New Delhi (CSIR-IGIB/IHEC/2020-21/01.)
Version history
- Received: February 1, 2021
- Accepted: June 7, 2021
- Accepted Manuscript published: June 9, 2021 (version 1)
- Version of Record published: July 19, 2021 (version 2)
Copyright
© 2021, Kumar et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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FnCas9 based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV2 variants on a paper strip
eLife 10:e67130.
https://doi.org/10.7554/eLife.67130
Further reading
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- Epidemiology and Global Health
Paxlovid, a SARS-CoV-2 antiviral, not only prevents severe illness but also curtails viral shedding, lowering transmission risks from treated patients. By fitting a mathematical model of within-host Omicron viral dynamics to electronic health records data from 208 hospitalized patients in Hong Kong, we estimate that Paxlovid can inhibit over 90% of viral replication. However, its effectiveness critically depends on the timing of treatment. If treatment is initiated three days after symptoms first appear, we estimate a 17% chance of a post-treatment viral rebound and a 12% (95% CI: 0%-16%) reduction in overall infectiousness for non-rebound cases. Earlier treatment significantly elevates the risk of rebound without further reducing infectiousness, whereas starting beyond five days reduces its efficacy in curbing peak viral shedding. Among the 104 patients who received Paxlovid, 62% began treatment within an optimal three-to-five-day day window after symptoms appeared. Our findings indicate that broader global access to Paxlovid, coupled with appropriately timed treatment, can mitigate the severity and transmission of SARS-Cov-2.
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- Epidemiology and Global Health
Background:
Circulating omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) have been associated with various chronic diseases and mortality, but results are conflicting. Few studies examined the role of omega-6/omega-3 ratio in mortality.
Methods:
We investigated plasma omega-3 and omega-6 PUFAs and their ratio in relation to all-cause and cause-specific mortality in a large prospective cohort, the UK Biobank. Of 85,425 participants who had complete information on circulating PUFAs, 6461 died during follow-up, including 2794 from cancer and 1668 from cardiovascular disease (CVD). Associations were estimated by multivariable Cox proportional hazards regression with adjustment for relevant risk factors.
Results:
Risk for all three mortality outcomes increased as the ratio of omega-6/omega-3 PUFAs increased (all Ptrend <0.05). Comparing the highest to the lowest quintiles, individuals had 26% (95% CI, 15–38%) higher total mortality, 14% (95% CI, 0–31%) higher cancer mortality, and 31% (95% CI, 10–55%) higher CVD mortality. Moreover, omega-3 and omega-6 PUFAs in plasma were all inversely associated with all-cause, cancer, and CVD mortality, with omega-3 showing stronger effects.
Conclusions:
Using a population-based cohort in UK Biobank, our study revealed a strong association between the ratio of circulating omega-6/omega-3 PUFAs and the risk of all-cause, cancer, and CVD mortality.
Funding:
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institute of Health under the award number R35GM143060 (KY). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
