Role of CRISPR in Coronavirus Research
Clustered regularly interspaced short palindromic repeats, widely known as CRISPR is a simple yet powerful technology adapted from a naturally occurring gene-editing system in bacteria. Since its first discovery, CRISPR brought a revolutionary change in the field of genetics because it is fast, cheaper, more accurate, and highly efficient than any of the existing gene-editing technologies.
Bacteria use CRISPR as a defensive mechanism against bacteriophages. CRISPR derived RNA, along with several Cas proteins, including Cas9 would target the viral DNA and cut them apart, disabling the virus. No one knew how this process looked like until 2017 when a paper published in Nature communications described the process for the first time.
The potential applications of CRISPR include improving crops, correcting genetic defects, treating and preventing the spread of diseases. Since the onset of novel coronavirus pandemic, scientists and researchers around the world are working round the clock to understand its origin and
develop diagnostics, vaccines, and treatments to contain its spread. There are many scientists among them who believe in CRISPR’ s potential to detect and prevent SARS-CoV-2 infection.
CRISPR Based Detection of SARS-CoV-2
Containing the spread of novel coronavirus has become the utmost priority of most countries. Testing, tracking, and social distancing is the strategy WHO recommends for containing the spread of COVID-19. It has become evident by now that widespread testing is crucial for controlling the pandemic with countries like South Korea flattening the curve effectively.
Currently, the RT-PCR test is the only reliable test available for detecting SARS-CoV-2. But the test has limitations like long turnaround time and limited availability of reagents and equipment. This has forced scientists to look for other methods to detect the virus. And this includes CRISPR based detection of SARS-CoV-2.
CRISPR Based DETECTR Assay
In the DETECTR assay, viral RNA is extracted and amplified by primers that target the N2 and E genes of the novel coronavirus. The assay then makes use of Cas12a-sgRNA to target and cleave the ssDNA, which would cleave the supplied fluorescently labeled ssDNA substrates. Upon cleavage, fluorescence can no longer be quenched. Lateral flow strips can be used to visualize this change in fluorescence. The test doesn’t take more than 45 minutes to complete and has shown a positive prediction rate of 95% and a 100% negative prediction rate. It is the fastest test developed so far to detect SARS-CoV-2.
CRISPR Based SHERLOCK Test
SHERLOCK test extracts RNA from the sample and subjects it to isothermal recombinase polymerase amplification to produce cDNA of S and Orf1ab genes of the viral RNA. Cas13 with crRNA is then used to detect the target sequence and perform a trans cleavage reaction that would cleave a nearby sequence that contains a fluorophore that is quenched when intact and released when cleaved. The fluorescence resulting from cleavage can be then visualized using lateral flow strips.