The potential of CRISPR-based diagnostic assays and treatment approaches against COVID-19

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In their review paper recently published in the Indian Journal of Clinical Biochemistry, a group of authors discusses currently available and forthcoming CRISPR-based diagnostic assays, as well as the possibility of using the CRISPR/Cas system as a salient treatment or prevention strategy in the fight against coronavirus disease 2019 (COVID-19).

The unfaltering COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a novel member from the already known betacoronavirus genus that shows 79% genetic similarity with the original SARS-CoV (as demonstrated by metagenomic next-generation sequencing).

Such escalating number of cases and deaths – despite the wide availability of the vaccines in our armamentarium – has left us searching for tools and approaches for swift, reliable, and inexpensive detection methods on the one hand, and new, ameliorated treatment strategies on the other.

When the diagnostic approach is concerned, the currently used reverse transcription-polymerase chain reaction (RT-PCR) has unquestionable utility but certain shortcomings. Furthermore, from the treatment side, remdesivir is the only antiviral agent fully approved for the treatment of COVID-19, but its utility has been debated.

Novel diagnostic tools, which are based on the clustered regularly interspaced short palindromic repeats / Cas (CRISPR-Cas) system, might be our answer to that problem. This complex system was initially identified in bacteria, where it confers innate protection against viral invasion; today, it keeps driving significant advances in the field of molecular technology in combination with newer genome editing tools.

How do CRISPR-based diagnostic/therapeutic methods work?

At the moment, tools for modifying genomes in experimental systems can be group into four classes: nucleases, transposases/recombinases, base editors and prime editors. The aforementioned CRISPR-Cas system includes both DNA- and RNA-targeting nucleases such as Cas9 and Cas13 enzymes, respectively.

In short, CRISPR-based diagnostic methods primarily depend on the concept that nucleic acids are valuable biomarkers for various diseases. This is achieved by identifying specific sequences associated with the infective virus. Then custom single guide RNA coupled with Cas nuclease can cleave a target specifically in order to generate a readable signal.

The relatively high specificity of single guide RNA targets enables the CRISPR system to differentiate between various strains of viruses. Another beneficial aspect of this technology is the use of simple reagents and point-of-care devices (e.g., the use of paper-based lateral flow assays).

Advanced diagnostic approaches with CRISPR/Cas

Two CRISPR-based methods may serve as potential diagnostic platforms for SARS-CoV-2: DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) method and the sensitivity enzymatic reporter unlocking (SHERLOCK) method. Importantly, the protocols explicitly outlined for SARS-CoV-2 detection can be made cheaply in an hour (or even less).

To overcome the obstacles faced with real-time RT–PCR-based assays, a combined isothermal amplification with CRISPR–Cas12 DETECTR technology has been developed to rapidly detect SARS-CoV-2 in clinical samples (i.e., in 30 or 40 minutes). More specifically, the DETECTR method aims to detect the presence of the nucleocapsid (N) and envelope (E) gene variants specific to SARS-CoV-2.

Consequently, a positive result is produced if both genes are detected, and the technique has been optimized to exclude false positives stemming from related coronaviruses. In addition, the time necessary for developing and validating DETECTR assay revealed that this technology could be swiftly mobilized to diagnose other emerging zoonotic viral infections.

Breakthroughs in treating COVID-19

Researchers have quickly adopted the CRISPR-Cas mechanism as a viable treatment strategy to amend genetic abnormalities in human cells as a way to treat diseases or as an indispensable tool in chemical biology. Considering it confers protective immunity against invading pathogens (such as bacteriophages), its use as a potential antiviral therapy emerged right from the start.

And of course, recently, the application of CRISPR/Cas13 was explored against the potential targets in the SARS-CoV-2 viral genome. Several structural, non-structural, and accessory proteins of the virus have been evaluated, which can be targeted by the CRISPR/Cas13 system with much more precision, sensitivity, and specificity.

One drawback of this strategy is the potential occurrence of CRISPR RNA target site mutations, which may lower its potency as viruses tend to modify their sequences in response to introduced treatment. Nonetheless, a straightforward alteration in the CRISPR RNA sequence may solve this problem and will further open the door for designing tools against other viruses.

In conclusion, the CRISPR-Cas system shows substantial promise in both diagnostics and treatment of COVID-19 and may as well change the trajectory of molecular diagnosis and precision medicine. For now, further research is needed to fully exploit its potential against SARS-CoV-2 and other potential viral threats.

Journal reference:
Dr. Tomislav Meštrović

Written by

Dr. Tomislav Meštrović

Dr. Tomislav Meštrović is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university - University North. In addition to his interest in clinical, research and lecturing activities, his immense passion for medical writing and scientific communication goes back to his student days. He enjoys contributing back to the community. In his spare time, Tomislav is a movie buff and an avid traveler.


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