SARS-CoV-2 detection using CRISPR diagnostics

September 11, 2021

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The scientific community has been racing against time since the day the WHO declared COVID-19 as a pandemic. Researchers around the globe began working almost immediately on SARS-CoV-2 detection techniques that would be affordable, effective and quick. Nucleic acid testing is the primary method of SARS-CoV-2 detection and diagnosis. A vast number of reverse transcription polymerase chain reaction (RT-PCR) kits have been designed all over the world to detect SARS-CoV-2 genetically. However, this method of testing has some drawbacks including incidents of false-negative results, shortage of kits, and a waiting period of several hours to obtain the test result. The RT-PCR method also requires costly equipment, a molecular testing laboratory and supervision by a trained scientist. Hence, the search for a rapid, accurate and simple method of detection is underway.

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In the last few years, CRISPR-based techniques have shown good promise for highly specific and sensitive molecular diagnostics. Therefore, CRISPR diagnostics have been of great interest for the detection of SARS-CoV-2 in the ongoing pandemic. CRISPR is short for ‘clusters of regularly interspaced short palindromic repeats.’ CRISPR technology is a tool used to edit genomes. It is simple, but powerful in its ability to alter DNA sequences and modify gene sequences.

There are two general steps in the process of detection using CRISPR : first, the CRISPR-RNA complex cuts the target region of the patient sample, and this activates the next step: collateral cleavage of the surrounding nucleic acids. CRISPR Cas12, Cas13, and Cas14 (CRISPR associated) effector proteins have a unique cleavage ability. They act like molecular scissors, which enables them to cleave surrounding nucleic acid once they bind to the target site.

A CRISPR based technique called SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking) has also been studied for the detection of SARS-CoV-2. This testing method starts with RNA purified from patient samples, similar to the PCR method.The SHERLOCK COVID-19 detection protocol works in three steps and can be completed in 1 hour, starting from nucleic acid extraction as used for qRT-PCR tests.

Step 1 – 25 min incubation period – isothermal amplification of the extracted nucleic acid sample using a commercially available recombinase polymerase amplification (RPA) kit;
Step 2 – 30 min incubation period – detection of pre-amplified viral RNA sequence using Cas13 effector protein;
Step 3 – 2 min incubation period – visual read out of the detection result by eye using a commercially-available paper dipstick.

There are various CRISPR-based techniques undergoing examination and refinement to be implemented as viable testing methods for SARS-CoV-2. While CRISPR-based techniques are faster and more sensitive than the traditional testing methods, it is important to make sure that CRISPR-based testing is accessible for countries where health infrastructure is not well-funded or well-developed. The current CRISPR-based methods need to be tested extensively against samples from different sources, such as blood, saliva, and nasopharyngeal specimens, in order to prove their specificity, sensitivity, and duration. They will also need to be tested on clinical samples of other coronaviruses to prove that they can distinguish between similar viruses despite the shared conserved genomic regions. Manufacturing the CRISPR proteins is also a hurdle to be tackled, since Cas13 and Cas14 proteins are currently not available commercially. This means that laboratories have to express and purify the proteins on-site, which is time consuming and defeats the purpose of switching to CRISPR-based testing.

Indian researchers like Dr. Debojyoti Chakraborty are also deeply interested and invested in CRISPR technology and gene editing technology. Gene editing research in India has been off to a good start and shows signs of a promising future. From India, The Tata CRISPR test is “the world’s first diagnostic test to deploy a specially adapted Cas9 protein to successfully detect the virus causing Covid-19.” The technique moved from R&D to a scalable and reliable test in less than 100 days. The Tata CRISPR test boasts accuracy levels similar to conventional RT-PCR tests, with quicker turnaround time, more affordable equipment, and greater ease of use.

Promega is a biotechnological supply company which has been actively working to supply and encourage research of testing kits as well as diagnostic techniques during COVID-19. Thermo Fisher Scientific supplies tools and solutions required to carry out the CRISPR genome editing process.

In conclusion, we see that CRISPR shows great potential to improve the detection and diagnosis process of SARS-CoV-2 and many other viruses. With the necessary improvements and modifications, CRISPR can revolutionise the diagnostic process and make things much easier for the world in its battle against viral diseases.

Learn more about the recent research in diagnosis, therapeutic interventions and analytical characterization of SARS-CoV-2 virus at Biosimilar Workshop 2021. Register at https://www.biosimilarworkshop.com/registration/

References:

  • https://www.frontiersin.org/articles/10.3389/fcimb.2021.663949/full#h7
  • https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf
  • https://www.iaea.org/newscenter/news/how-is-the-covid-19-virus-detected-using-real-time-rt-pcr
  • https://pib.gov.in/PressReleasePage.aspx?PRID=1656770
  • Written by: Ria Gada