Possibility of CRISPR-based diagnostic assay and therapeutic approach for COVID-19

In their recently published review paper Indian Journal of Clinical Biochemistry, The authors’ group discusses the potential for using the CRISPR / Cas system as a prominent therapeutic or preventive strategy in the fight against coronavirus disease, as well as currently available and upcoming CRISPR-based diagnostic assays 2019 (COVID- 19).

The unwavering COVID-19 pandemic is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It is a new member of the known betacoronavirus genus, showing 79% genetic similarity to the original SARS-CoV (according to (proven) metagenomic next-generation sequencing).

This increasing number of cases and deaths, on the one hand, provides tools and approaches for fast, reliable and inexpensive detection methods, despite the widespread availability of vaccines in our arsenal. On the other hand, they are forced to seek new and improved treatment strategies.

When it comes to diagnostic approaches, the currently used reverse transcription-polymerase chain reaction (RT-PCR) has undoubted utility, but it has certain drawbacks. In addition, on the therapeutic side, remdesivir is the only fully approved antiviral agent for the treatment of COVID-19, but its usefulness is being debated.

A new diagnostic tool based on a clustered, regularly spaced short palindrome repeat / Cas (CRISPR-Cas) system may be our answer to that problem. This complex system was first identified with bacteria that provide innate protection against viral invasion. Today, it continues to drive significant advances in the field of molecular technology in combination with new genome editing tools.

How do CRISPR-based diagnostics / treatments work?

At this time, tools for modifying the genome in systems can be divided into four classes: nucleases, transposases / recombinases, base editors, and prime editors. The aforementioned CRISPR-Cas system contains both DNA and RNA targeting nucleases, such as Cas9 and Cas13 enzymes, respectively.

In short, CRISPR-based diagnostic methods rely primarily on the notion that nucleic acids are valuable biomarkers for a variety of diseases. This is achieved by identifying specific sequences associated with the infectious virus. The custom single-guide RNA combined with Cas nuclease can then cleave the target specifically to generate a readable signal.

Due to the relatively high specificity of single-guided RNA targets, the CRISPR system can distinguish between different viral strains. Another beneficial aspect of this technology is its simple use. reagent And point of care equipment (eg, use of paper-based immunochromatography).

Advanced diagnostic approach with CRISPR / Cas

Two CRISPR-based methods may serve as potential diagnostic platforms for SARS-CoV-2. The CRISPR transreporter (DETECTR) method and the sensitive enzyme reporter unlock (SHERLOCK) method targeting DNA endonucleases. Importantly, the protocols explicitly outlined for SARS-CoV-2 detection can be inexpensively created in an hour (or less).

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

As a result, a positive result is generated when both genes are detected, and the technique is optimized to rule out false positives due to association. Coronavirus.. In addition, the time required to develop and validate the DETECTR assay has revealed that this technology can be rapidly mobilized to diagnose viral infections of other zoonotic diseases.

Breakthrough in the treatment of COVID-19

Researchers quickly adopted the CRISPR-Cas mechanism as a viable therapeutic strategy for correcting genetic abnormalities in human cells, either as a method of treating disease or as an essential tool in chemical biology. .. Its use as a potential antiviral therapy has emerged from the beginning, given that it provides protective immunity against invading pathogens (such as bacteriophage).

And, of course, recently, CRISPR / Cas13 applications have been investigated for potential targets in the SARS-CoV-2 viral genome. Several structural, non-structural, and accessory proteins of the virus have been evaluated and can be targeted with much higher accuracy, sensitivity, and specificity by the CRISPR / Cas13 system.

One drawback of this strategy is the potential for mutations in the CRISPR RNA target site. This can reduce its potency as the virus tends to sequence in response to the treatment in which it was introduced. Nonetheless, direct changes in the CRISPR RNA sequence may solve this problem, further opening the door for designing tools against other viruses.

In conclusion, the CRISPR-Cas system holds great promise for both diagnosis and treatment of COVID-19 and may change the course of molecular diagnosis and precision medicine. For now, further research is needed to take full advantage of its potential for SARS-CoV-2 and other potential viral threats.