|Year : 2020 | Volume
| Issue : 4 | Page : 302-304
FNCas9 editor-linked uniform detection assay: An innovative COVID-19 sleuth
Tarun Kumar Suvvari, MD Nawaz, Mani Kruthika Mantha
Rangaraya Medical College, Kakinada, Andhra Pradesh, India
|Date of Submission||14-Nov-2020|
|Date of Acceptance||24-Nov-2020|
|Date of Web Publication||30-Dec-2020|
Mr. Tarun Kumar Suvvari
Rangaraya Medical College, Kakinada, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Relying on traditional diagnostic approaches like quantitative polymerase chain reaction is not appropriate for both time and cost in sudden outbreaks and pandemics such as COVID-19. In such cases, diagnostic approaches through rapid sequencing of pathogenic DNA and RNA are important for accelerated clinical prognosis. FNCas9 Editor-Linked Uniform Detection Assay (FELUDA) is one such diagnostic approach toward the pathogenic genome, developed by CSIR-IGIB. FELUDA is a paper strip diagnostic test like pregnancy, based on clustered regularly interspaced short palindromic repeats/Cas technology of gene editing. FELUDA showed 96% sensitivity and 98% specificity across all range of viral loads in clinical samples. Due to its low cost, it can drastically reduce the Indian budget toward the rapid testing and COVID-19 diagnostic kits.
Keywords: Clustered regularly interspaced short palindromic repeats, COVID-19, FNCas 9, FNCas9 Editor-Linked Uniform Detection Assay, severe acute respiratory syndrome-CoV-2
|How to cite this article:|
Suvvari TK, Nawaz M D, Mantha MK. FNCas9 editor-linked uniform detection assay: An innovative COVID-19 sleuth. Biomed Biotechnol Res J 2020;4:302-4
|How to cite this URL:|
Suvvari TK, Nawaz M D, Mantha MK. FNCas9 editor-linked uniform detection assay: An innovative COVID-19 sleuth. Biomed Biotechnol Res J [serial online] 2020 [cited 2022 Aug 16];4:302-4. Available from: https://www.bmbtrj.org/text.asp?2020/4/4/302/305642
| Introduction|| |
COVID-19 caused by severe acute respiratory syndrome (SARS)-CoV-2 led to a new public health crisis, and public health emergency was declared on January 30, 2020. The outbreak was declared a pandemic on March 11, 2020, by the World Health Organization (WHO). The primary mode of spread for COVID-19 is respiratory droplets produced from an infected person's coughs or sneezes. The SARS-CoV-2 targets the angiotensin that converts enzyme 2 receptors and binds to them, commonly present in the lung, kidney, and gastrointestinal tract.
The strategy to control the spread of the virus, as advised by the WHO, requires testing all suspected individuals, isolating them if they test positive, and tracing all their contacts to repeat the process. However, in densely populated countries, India, this strategy can be possible only when we meet the testing requirements.
| Problem with COVID-19 Diagnostics|| |
Public health experts and scientists have expressed concern about the need for increased testing capacity for the COVID-19, and some governments have taken extraordinary measures to increase the availability of testing. Currently, nucleic-acid-based tests are giving high sensitivity and early detection of COVID-19.
However, the “gold standard” test (Reverse transcription-polymerase chain reaction [RT-PCR]) takes several hours to complete and requires extensive human labor, equipment like RNA extraction kits, and quantitative-PCR machines, which are shortage in most places frequently. It requires huge efforts to increase COVID-19 testing into large numbers. There is a need for urgent evaluation of alternative methods, testing agents, and techniques to permit nucleic acid testing during the shortage of COVID -19 testing.
To overcome these difficulties, rapid antigen tests (RATs) have been designed, but these are found to be not accurate to detect coronavirus. However, due to scarcity of equipment and lack of feasibility, RATs have become a major part of testing in many rural and some parts of urban areas. Due to this low sensitivity and specificity of RATs, many cases are becoming undiagnosed or else landing in severe conditions, which is a major concern for the government and medical fraternity to control the pandemic.
Clustered regularly interspaced short palindromic repeats Cas9 gene editing technology and its application to COVID-19 diagnostics
Clustered regularly interspaced short palindromic repeat (CRISPR) components easily detect various nucleic acid targets from a wide variety of pathogens. The evolution of CRISPR Cas9 usage for biosensing nucleic acids led to the innovation of successful diagnostic tools during the COVID-19 pandemic. CRISPR proteins can directly bind to target DNA/RNA and undergo conformational changes, which lead to the cleavage of targets generating a reporter-based signal outcome. Accuracy of DNA inquisition, successive enzyme activity, and the huge diversity of targets are immensely needed to make clinical decisions. Recent advances using CRISPR components for nucleic acid detection can identify the target using substrate cleavage mediated by an active CRISPR ribonucleoprotein (RNP) complex or by binding through a catalytically inactive RNP complex.
Cas is a family of proteins that include cellular apoptosis susceptible proteins. SpCas 9 is the widely used Cas9, which is derived from Streptococcus pyogenes. FNCas9 is derived from the bacterium Francisella Novidium, which shows a negligible binding affinity to off targets, rendering it highly specific in target recognition. FnCas9 can direct both homology-directed repair (HDR) and nonhomologous end-joining–mediated DNA repair, generates a higher rate of HDR and negligible off targeting. FnCas9 produces staggered cleavage, higher HDR rates, and very low nonspecific genome editing compared to SpCas9. FNCas9 is 100% accurate in single-nucleotide variants.
| FnCas9 Editor-Linked Uniform Detection Assay – A Sleuth|| |
FnCas9 Editor-Linked Uniform Detection Assay (FELUDA) utilizes a direct Cas9-based enzymatic readout for recognizing nucleotide sequence and distinguishing nucleobase personality without the prerequisite of trans-cleavage activity. FELUDA was extremely precise in detecting single-nucleotide variants, including heterozygous carriers of a mutation. FELUDA is semi-quantitative, which can acclimatize multiple signal detection platforms. FELUDA showed 96% sensitivity and 98% specificity across all viral loads in clinical samples.
Azhar et al. have proposed a prototype of FELUDA that can be adapted to a point-of-care or home testing assay. It is a PCR machine free version of FELUDA using recombinase polymerase amplification (RPA) that can detect SARS-CoV2 RNA in biological samples within 30 min. They have successfully developed a 15-min benchtop method for RNA extraction from saliva and an on-body 30-min RPA-FELUDA (tested using synthetic RNA fragments), thus generating an end-to-end instrumentation free testing protocol.
| Tata Medical and Diagnostics Limited Check|| |
CSIR-IGIB developed FELUDA, and Tata Medical and Diagnostics Ltd. (TataMD) has partnered with them and launched TataMD CHECK (new diagnostic testing for COVID-19) on November 9, 2020. TataMD Check was approved by the Indian Council of Medical Research and Drug Controller General of India, and it will be released into the market soon. TataMD CHECK is a paper strip-based test which is more efficient and simpler with an image-based visual result readout. The total testing time from RNA-extracted samples in the laboratory to result is 75 min only with a fast reaction time of 45 min–50 min in the laboratory.
TataMD CHECK provides high-quality, quick results using quality equipment, and minimally trained staff allow for quick adoption and scale with the potential to reach rural parts of the country.
Health technology assessment of FnCas9 Editor-Linked Uniform Detection Assay
Among the three diagnostic tests such as FELUDA, RT-PCR, and RAT, HTA of FELUDA showed the most cost-effective testing strategy at INR 500 per test, which has the potential to improve India's testing capacity.
At the present need for testing and market price, FELUDA would bring down the country's testing cost by almost one-thirs of the present and save around INR 800 million (approximately USD 11 million) per day. The shorter result time, ease of doing the test, and analysis would increase testing availability and enhance the testing rates in India.
Although Lateral Flow Assay gives semi-quantitative readout, for the accurate and sensitive diagnosis, a large number of primers and sgRNAs are tested for strong readout, and through this, they can minimize off targeting.
- Only two genes (N and S) of the 21 targeted regions satisfied the criteria of FELUDA
- Single-gene FELUDA done on frozen samples compared with qRT-PCR is less sensitive than double-gene FELUDA on frozen samples.
- For consistent results, optimized conditions and high-quality PCR reagents are necessary.
| Conclusion|| |
FELUDA is a pathbreaking innovation in the field of COVID-19 diagnostics, which is essential during this pandemic. Due to its low price, high sensitivity, and specificity across a wide range of viral loads, it would boost India's testing capacity.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Suvvari TK, Kutikuppala LV, Babu GK, Jadhav M. Understanding the unusual viral outbreak: Coronavirus disease 2019. J Curr Res Sci Med 2020;6:3-10. [doi: 10.4103/jcrsm.jcrsm_30_20].
Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R. Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection. RNA 2020;26:771-83.
Ravi N, Cortade DL, Ng E, Wang SX. Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape. Biosens Bioelectron 2020;165:112454.
Vandenberg O, Martiny D, Rochas O, van Belkum A, Kozlakidis Z. Considerations for diagnostic COVID-19 tests. Nat Rev Microbiol 2020. doi: 10.1038/s41579-020-00461-z.
Xiang X, Qian K, Zhang Z, Lin F, Xie Y, Liu Y, et al
. CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia. J Drug Target 2020;28:727-31.
Chekani-Azar S, Gharib Mombeni E, Birhan M, and Yousefi M. CRISPR/Cas9 gene editing technology and its application to the coronavirus disease (COVID-19), a review. J Life Sci Biomed 2020;10:1-9.
Azhar M, Phutela R, Kumar M, Ansari AH, Rauthan R, Gulati S, et al
. Rapid, accurate, nucleobase detection using FnCas9. medRxiv 2020. [pre-print]. [doi: 10.1101/2020.09.13.20193581].
Mukherjee K. HTA of FELUDA diagnostic test for COVID-19. Cambridge Open Engage 2020. [doi: 10.33774/coe-2020-ztz0t].