Student contributors – Rahul Dutta, Shaurav Bhattacharyya, Aniket Das ( 3^rd year B. Tech Biotechnology)

            The World is witnessing an era where the tiniest being, a mere virus, has taken control over the so-called intelligent minds. Is this the curse of the Almighty or the action of the Evil is not known but what is known is that we must win over this battle to save the innocent human hearts who are becoming victims in the hands of this deadly virus. In recent years, the world is seeing a surge in large-scale epidemics from emerging viruses (like MERS, Ebola & the present SARS-CoV-2) and it is seen that the initial zoonotic animal-to-human transmission event is involved significantly in all of these epidemics. Each time, the viral threat is glorified by the lack of rapid diagnostic testing and quick treatments to obstruct the spread thus hindering public health. The scientists and researchers at every corner of the globe are working relentlessly to find a solution at least to curtail the steep progressive curve of the no. of deaths until the proper vaccine is successfully developed. Here, we are presenting some of the directions in which the scientists are trying to find a way to combat the present crisis scenario.

Utility of BCG Vaccination:

            Scientists have found that heterologous protection conferred by the BCG vaccine could also be effective in patients who have contracted the COVID-19 infection. This is evident from the fact that higher mortality rates are observed in those regions not having universal BCG vaccination policies like Italy, the USA, etc. The protection that the BCG vaccine is conferring can be due to its non-specific mode of action. This means that molecular similarity between BCG and other viral antigens could have led to the formation of some memory B and T cells which can recognize both BCG and other respiratory pathogens. It has been also investigated that BCG could have stimulated the epigenetic reprogramming of the innate immune cells. The monocytes may have undergone modification at the promoter sites of the genes encoding inflammatory cytokines which are now resulting in a more active immune response as those modified monocytes have been reactivated & triggered by the coronavirus antigen in those who have received the BCG vaccination previously.

Applicability & future of Nanotechnology:

            Today nanotechnology or the science of nanoscale structures is one of the most interesting branches of science. A new nanomedicine developed at the SN Bose National Centre for Basic Sciences (SNBNCBS) in Kolkata, has the potential in the treatment of Covid-19. Our body continuously adds and removes oxygen through oxidation and reduction (redox) reactions. However, reactive oxygen species (ROS) free radicals are also generated from these processes. During the viral or bacterial attack, ROS, or oxygen stress level of our body is naturally generated by our immune cells so that they can efficiently perform their functions. In the new nanomedicine formulation, nanoparticles extracted from manganese salt are combined with the citrus extract. Clinical trials on mice have shown that the new nanomedicine adds up the oxidative stress in a controlled manner and thus finds a potential application in controlling virus infections like the Covid-19. Another interesting application of nanomedicine is the use of silver nanoparticles. Colloidal silver or a suspension of microscopic, electrically charged particles of silver bear a wide potential to act against almost 650 different kinds of pathogens which include bacteria, fungus, and viruses. They aid in inhibiting the oxygen metabolizing enzymes in these pathogens. Covid-19 infections start mildly in the upper respiratory airways. Hence it can be applied through inhalation delivery methods to suppress the proliferation of the virus during the early days of infection.

Remarkable potential of CRISPR-based diagnostics & therapeutics:

            CRISPR (clustered regularly interspaced short palindromic repeats) is referred to as a family of DNA sequences that are exclusively found within the genetic makeup of prokaryotic organisms. These sequences are derived from DNA fragments of some previously attacking bacteriophages. The CRISPR sequences whenever they encounters any complementary DNA sequence concerning it, they bind to that and the Cas9 (CRISPR-associated protein 9) nuclease-type enzyme associates with the CRISPR sequences & cleaves those specific strands of DNA. Thus, whenever, any future invasion of the same bacteriophage occurs, this phenomenon inactivates the invading DNA of the attacking bacteriophage thus conferring the prokaryotes with a mode of acquired immunity. This technique can be harnessed and applied in viral and pathogen rapid diagnostic and therapeutic purposes. Cas13 nuclease-type enzymes are RNA-targeting proteins by virtue of which property, these nucleases can degrade specific target RNAs without producing any effect to the host genome. This property makes Cas13 nucleases an interesting candidate for study. On the other hand, Cas12 type nucleases work on DNA. Both Cas12 and Cas13 nucleases can also cleave other nucleic acid molecules (except the target ones) present in the vicinity of their target site. This trait (called the trans or collateral cutting activity) can be applied to CRISPR diagnostics where the non-targeted nucleic acids can be utilized to make certain color-tagged probes for detection purposes.

            Feng Zhang’s group first reported a CRISPR-based nucleic acid detection technique called SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing). Zhang and his team identified two genes (S gene and Orf1ab) from the SARS-CoV-2 genome as their targets. In the SHERLOCK protocol for coronavirus detection, firstly, the synthetic viral RNA is amplified by applying the recombinase polymerase amplification (RPA) technology. This process is followed by in vitro transcription where the amplified DNA gets converted to RNA. Then, RNA detection is done using Cas13 nuclease and specific crRNA (gRNA) targeting specific sequences. Finally, the visual color readout is done where the cleaved reporter RNA with labeled ends are captured on specific antibody bands (to form the visual test band) on lateral flow strips. This protocol has been demonstrated to detect coronavirus RNA from patient samples in very less time (almost less than an hour) and here no special instrumentation is required (as isothermal signal amplification process proceeds through a constant temperature thus not requiring the thermocycler as required by the qRT-PCR technique).

            Mammoth Biosciences proposed a new method which is DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) assay which recruits Cas12a for sensitive DNA detection purposes. The method utilizes the same CRISPR-based detection of the two genes (N-gene and E-gene) which, by bioinformatics computational methods, are found to be conserved in SARS-CoV-2 genome. This method utilized the technique of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for amplifying the RNA strands to generate a huge number of DNA strand loops and this method takes about 30 minutes which is less when compared to the SHERLOCK-based protocol. This is because here, no extra time is required to be spent on the additional IVT steps. Such a rapid diagnostic platform would be particularly valuable in high-risk areas such as airports, clinics, and hospitals. 

            Other than detecting, a new CRISPR-based antiviral strategy targeting the SARS-CoV-2 virus appears to effectively degrade RNA (as has been proposed in a Stanford paper). This aids in killing or knocking out the virus which can thus be subsequently applied to the therapeutic sector. This approach, termed PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), employs six CRISPR RNAs that can target 91 percent of the 3,051 sequenced coronaviruses, thus signifying its broad coverage. This CRISPR-based approach recruits Cas13d type RNA endonuclease which employs customizable CRISPR-associated RNAs. The reason for choosing Cas13d over other Cas13 proteins is because it has a small size, is highly specific in action, and possesses a strong catalytic activity. Cas13d is made to target & destroy two conserved sites of the SARS-CoV-2 viral genome, which encode the RdRP and Nucleocapsid proteins, known to be essential for coronavirus replication and function. This technique now needs to be tested & validated with live SARS-CoV-2 viruses and whether they are having any off-target side effects must be thoroughly evaluated via clinical screening. An effective and safe in vivo delivery method into the human respiratory tract cells must be developed. The remarkably great potential of the CRISPR-based therapeutic systems lies in the fact that once the genetic targets of a new virus are identified, altering a previous treatment becomes simple which can be quickly implemented during the birth of any future pandemics.

Conclusion:

Hence, it can be inferred from the above discussion that the BCG vaccination strategy, the application of nanomedicines, and the CRISPR-based diagnostic & therapeutic systems hold a great promise & a bright future to emerge out victoriously against the threat of Covid-19.

References

• Broughton, J.P., Deng, X., Yu, G. et al.CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0513-4
• Redelman-Sidi, G. Could BCG be used to protect against COVID-19?. Nat Rev Urol(2020). https://doi.org/10.1038/s41585-020-0325-9