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.
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.)
Reviewing Editor
- Yamuna Krishnan, University of Chicago, United States
Publication 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
Background: Home-based self-sampling for human papillomavirus (HPV) testing may be an alternative for women not attending clinic-based cervical cancer screening.
Methods: We assessed barriers to care and motivators to use at-home HPV self-sampling kits during the COVID-19 pandemic as part of a randomized controlled trial evaluating kit effectiveness. Participants were women aged 30-65 and under-screened for cervical cancer in a safety-net healthcare system. We conducted telephone surveys in English/Spanish among a subgroup of trial participants, assessed differences between groups, and determined statistical significance at p<0.05.
Results: Over half of 233 survey participants reported that clinic-based screening (Pap) is uncomfortable (67.8%), embarrassing (52.4%), and discomfort seeing male providers (63.1%). The last two factors were significantly more prevalent among Spanish versus English speakers (66.4% vs. 30% (p=0.000) and 69.9 vs. 52.2% (p=0.006), respectively). Most women who completed the kit found Pap more embarrassing (69.3%), stressful (55.6%), and less convenient (55.6%) than the kit. The first factor was more prevalent among Spanish versus English speakers (79.6% vs. 53.38%, p=0.001) and among patients with elementary education or below.
Conclusions: The COVID-19 pandemic influenced most (59.5%) to participate in the trial due to fear of COVID, difficulty making appointments, and ease of using kits. HPV self-sampling kits may reduce barriers among under-screened women in a safety-net system.
Funding: This study is supported by a grant from the National Institute for Minority Health and Health Disparities (NIMHD, R01MD013715, PI: JR Montealegre).
Clinical trial number: NCT03898167.
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- Epidemiology and Global Health
Local cervical cancer epidemiological data essential to project the context-specific impact of cervical cancer preventive measures are often missing. We developed a framework, hereafter named Footprinting, to approximate missing data on sexual behaviour, human papillomavirus (HPV) prevalence, or cervical cancer incidence, and applied it to an Indian case study. With our framework, we (1) identified clusters of Indian states with similar cervical cancer incidence patterns, (2) classified states without incidence data to the identified clusters based on similarity in sexual behaviour, (3) approximated missing cervical cancer incidence and HPV prevalence data based on available data within each cluster. Two main patterns of cervical cancer incidence, characterized by high and low incidence, were identified. Based on the patterns in the sexual behaviour data, all Indian states with missing data on cervical cancer incidence were classified to the low-incidence cluster. Finally, missing data on cervical cancer incidence and HPV prevalence were approximated based on the mean of the available data within each cluster. With the Footprinting framework, we approximated missing cervical cancer epidemiological data and made context-specific impact projections for cervical cancer preventive measures, to assist public health decisions on cervical cancer prevention in India and other countries.
