A novel, quicker and economical ‘Dip-strip’ methods of Covid-19 detection based on CRISPR technology: The limitless scope of research in the Biotechnology sector
Student Contributor: Sayantan Das & Srijit Bera (B.Sc Biotechnology Sem-VI)
The landscape of Covid-19: Since December 2019, the Covid-19 has affected more than 3.5 million individuals globally with a case-fatality rate of more than 6% (data taken up to 30th April 2020) reported. Experience from China, Italy, UK, France, and the USA demonstrated that Covid-19 can overwhelm even the healthcare capacities of well-resourced countries where man-to-man disease transmission has been reported with mild or no symptoms.
The challenges: The biggest problem is that the virus is evolving continuously and several varieties have already been identified by scientists all over the world. Since there were no gold standard medicinal treatments available to date, interventions have focused only on contact tracing, quarantine, and physical distancing.
Since the initial pandemic wave, few countries like India have implemented physical distancing measures as a mandate according to the China model. Furthermore, in each time this emerging viral threat has hindered the public health response due to a lack of rapid, accurate, and accessible molecular diagnostics. However, whether the virus can potentially be transmitted in other ways than by respiratory droplets is still unknown to scientists.
Present detection: This has to be noted that this novel coronavirus (SARS-CoV-2) is a positive-stranded RNA virus. Hence, the Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of nasopharyngeal or oropharyngeal swabs typically has been used to confirm the presence of this virus and recommended by several laboratories, including the CDC and WHO. RT-PCR tests are rather quick, sensitive and can produce results in 3 to 4 hours, although this usually takes much longer time when samples are to be sent to specialized external laboratories (6-8 hours on average).
Though serological tests are fast and need minimal equipment but their usefulness might be limited for diagnosis of acute SARS-CoV-2 infection as it may take a couple of days to weeks following the start of symptoms in patients to get a measurable antibody response. The Rapid Diagnostic Test (RDT) is an immunoassay test that detects viral proteins (antigens) (Figure 1). Though this technique is fast but evidentially, it gives negative results for some cases. Moreover, RDTs can be damaged by heat and humidity, so an RDT should not be removed from its sealed packet until right before you are ready to use it otherwise it may show an invalid (false) result. Hence, there was an urgent need globally to develop a rapid testing tool for diagnostics.
The alternative: Among all the temporary procedures for virus detection, CRISPR based detection of microbial DNA/RNA has provided a quicker and economical alternative to RDT and RT-PCR. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a type of DNA sequence found in bacteria when they are previously infected by a specific variety of viruses called ‘bacteriophages’ to reproduce. Hence, these sequences are considered to be the anti-phage (i.e. antiviral) defense system of bacteria (or immune system for bacteria against phage viruses). The CRISPR mechanism relies on the guide RNA finding its complementary target sequence and the commonly used Cas9 nuclease (a type of molecular scissors) is cutting at the precise site. Together with CRISPR-associated proteins (or Cas), this “CRISPR-Cas” cutting-edge technology has been stated using to edit genes within organisms, development of biotechnology products, correcting genetic defects, improving crops, detection, and treatment of diseases and many more with immense scope of research for biotechnologists in India and abroad.
The DETECTIVES for Covid-19 detection:
- The SHERLOCK Technology for Coronavirus Detection:
In 2017, Feng Zhang’s group from the USA first reported a CRISPR-based nucleic acid detection technique called SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing). In their latest research came in February 2020, they targeted two genes (S gene and Orf1ab) from the SARS-CoV-2 genome.
The SHERLOCK detection technique for Covid-19 detection involves three steps:
- Firstly, the amplification of the viral RNA using recombinase polymerase amplification (RPA) technique followed by in vitro transcription of amplified DNA back into RNA.
- Secondly, they detected viral RNA using Cas13 nuclease and Synthego-supplied crRNA (CRISPR RNA) targeting specific sequences.
- Finally, they could read out the visual color change using a commercially-available paper dip-strip, which captures the cleaved RNA with labeled ends on specific antibody bands.
These two nucleases (Cas12a and Cas13a) have become popular in CRISPR diagnostics while Cas12a is DNA-specific, Cas13a works with RNA. An interesting feature of Cas12 and Cas13 nucleases is that they showed collateral cutting activity. On finding the target, the nucleases will cut other non-targeted nucleic acid molecules in the vicinity as well.
The researchers demonstrated that they could use this protocol to detect coronavirus RNA sequences with a sensitivity of 10-100 copies/microliter of the input sample. They note that this technology can be used for testing RNA purified from patient samples in ‘less than an hour’ without the need for special instrumentation like real-time PCR.
- The DETECTR Technology for Coronavirus Detection:
In 2018, Mammoth Biosciences reported a new method named DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter), for sensitive DNA detection using Cas12a nuclease. In March 2020, Mammoth Biosciences published a report on how the DETECTR platform could be adapted to detect SARS-CoV-2 RNA in just 30 minutes. Such a rapid diagnostic platform would be particularly valuable in high-risk areas.
The DETECTR detection protocol for Covid-19 virus detection involves following steps:
- Amplification of the RNA extracted from the sample using reverse transcription loop-mediated isothermal amplification (RT-LAMP) technique followed by Cas12a-based detection for viral N gene and E gene.
- Detection of DNA using Cas12a and Synthego-supplied crRNA targeting specific sequences.
- Visual readout using commercially available dip-strips. In the lateral flow-strip assay, a positive result will detect at least one of the two SARS-CoV-2 viral gene targets (N gene or E gene).
Comparing their methods to the SHERLOCK-based protocol, the Mammoth Biosciences team noted that their platform enabled faster detection (30 instead of 60 minutes) as they saved the time spent on the additional IVT (in vitro transcription) steps required for Cas13a-based detection. These techniques could detect Covid-19 much faster than the conventional RT-PCR technique (Figure 2).
Figure 2: Comparison between these three techniques for the detection of SARS-CoV-2
Where we are?
India had always nurtured creative research minds who always came up with a solution in the time of crisis. Recently, the scientists at the CSIR-IGIB have also developed a quicker (take less than 40 minutes for detection) and economical (~Rs. 500/- per sample detection) with high accuracy CRISPR based method that will not require any expensive machines for detection of SARS-CoV-2. They named their invention “FELUDA”, the detective character introduced by the legendary filmmaker Satyajit Ray. The CRISPR-FnCas9 based Dip-strips tests have been developed by the team members of Dr. Debojyoti Chakraborty and Dr. Souvik Maity as a simpler way of detecting SARS-CoV-2.
“Necessity is the Mother of Invention”.
In conclusion, I must say that in India, both in the government and private sectors the scope of scientific research in the biotechnology field is immense, and probably the sky is the limit. There is an immense need for research professionals in this fast-growing field with great scope for carrier growth.
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