PAC-MAN: The Coronavirus Killer
A CRISPR-mediated Coronavirus Treatment and Prevention Strategy
In mid-February, this year, Timothy Abbott, PhD Candidate at Stanford University’s Bioengineering Department, and Dr Marie La Russa, a research scientist working at Dr Stanley Qi’s lab at Stanford, found a way to create a therapeutic agent to prevent all forms of coronavirus.
In this breakthrough research, Abbott was using an approach called PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells) to attack RNA viruses, which includes the coronavirus family, and degrade their genetic code. The approach utilized a gene-editing technology known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which was specifically engineered to find and destroy the genetic sequences of SARS-CoV-2 (the novel coronavirus) and Influenza A viruses, both of which have a history of causing pandemics.
This came after researchers, including Qi, Abbott and Russa, identified the need for a pan-coronavirus vaccine/prevention strategy, because of the large potential of members of the coronavirus family, and other RNA viruses, to cause epidemics or pandemics. In fact, notable members of the family include the first SARS-CoV virus, responsible for the 2003 coronavirus outbreak that infected 8,098 people and claimed 774 lives worldwide, and the MERS-CoV virus still ravaging the Middle East, with a case count of 2,494 and 858 deaths.
And, of course, it includes the SARS-CoV-2 virus responsible for the current pandemic. The virus has caused economic shutdowns around the world, and in many countries like the United States and Canada, residents have been in lockdown for 4–6 months, as they wait for the situation to improve. And since the outbreak of the virus, around 13 million people have been infected and more than 573,000 lives have been claimed worldwide (at the time of writing this article on July 13, 2020).
But the reason coronaviruses are particularly notorious for causing pandemics and epidemics is their ability to evolve rapidly. Coronaviruses, as I mentioned earlier, are a type of RNA virus, meaning that they encode their genetic information in RNA (ribonucleic acid) instead of DNA. However, RNA replication, specifically, is extremely error-prone, because of the lack of error correction mechanisms. This means that the genetic code governing the structures of the coronavirus is constantly mutating and the virus is constantly evolving. This allows the virus to not only quickly evolve resistance to medications and sidestep immunity gained from vaccines meant to target an older version, but also grants them the ability to easily transfer between different species and adapt to new hosts, letting them “spontaneously transfer to humans from diverse animal hosts that act as viral reservoirs”, according to Abbott’s paper.
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How Does PAC-MAN Work?
The antiviral approach designed in this experiment relied on, as I mentioned before, a CRISPR-based system to recognize and degrade the viral genome once it is inserted into the cell, and the resulting viral mRNAs produced through RNA replication.
CRISPR consists of two main components that guide its functions and targets. The first is a guide RNA (gRNA) molecule that functions as a molecular GPS of sorts, guiding the CRISPR system to the correct site on the DNA, or the target RNA floating in the cell. This guide RNA contains a specific section known as the CRISPR-associated RNA (crRNA), which is the custom sequence that identifies the target. The other part of CRISPR is known as a Cas enzyme, which plays a role in cutting the genetic code once it reaches the target, like a pair of molecule scissors of sorts. This could be to allow insertion or deletion of a segment of the genetic code, or it could be used to completely degenerate specific RNA molecules, with the specific function based entirely on the type of Cas enzyme used.
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PAC-MAN uses a type of Cas enzyme known as Cas13d, which employs crRNAs to direct itself to specific RNA molecules in the cell, after which it carries out targeted RNA degradation. PAC-MAN uses this as a mechanism for targeting the SARS-CoV-2 genome for degradation, inhibiting viral gene expression.
However, even with the extremely specific and targeted CRISPR system, one problem remains: the coronavirus genome is still rapidly changing. The CRISPR system requires a crRNA strand that accurately identifies a specific molecule, which isn’t possible if the section of the genome identified is constantly mutating.
To work around this, Abbott used bioinformatics tools to identify regions of the virus that are highly conserved across all strains and types. This way, the crRNA strands used to target the SARS-CoV-2 genome is more effective because mutations in these regions are extremely rare. With this approach, the researchers found a group of just 6 crRNA sequences capable of targeting 91% of sequenced coronaviruses, and found that a cocktail with just 22 crRNAs is capable of targeting every sequenced coronavirus.
Putting PAC-MAN To The Test
Since the team was unable to access live versions of the SARS-CoV-2 virus at the time of the experiment, the approach was instead tested on synthesized fragments of the coronavirus genome, and with live infection of an Influenza A virus (IAV).
The team tested PAC-MAN by first developing a human epithelial cell line expressing the Cas13d gene and introduced the template crRNAs. Upon infection of these cell lines by both the SARS-CoV-2 synthesized genome fragments and the IAV virus, the researchers observed that the Cas13d systems in the cells were capable of effectively repressing expression and survival of the genomes in the cells, with high rates of targeted degradation.
This demonstrated that the Cas13d system was effective at targeting and cleaving RNA virus genomes in these cells upon infection. If administered as a therapeutic into the bodies of humans, this could prevent viruses from being able to infect cells in the first place, stopping the infection before it has even started.
But, despite its high potential as a vaccine against RNA viruses like SARS-CoV-2, two problems, true of all genetic engineering products, remain. It is still extremely unsafe to introduce CRISPR systems like the one used in this study into the human body. The systems are still not accurate enough to target just the viral genomes. Though they might be effective and fairly safe in the tissue cultures where it was tested in this study, it can have extremely harmful side effects if introduced into the body.
And the second issue lies in the fact that the current ways of introducing CRISPR into the body are extremely inefficient, meaning that only a small subpopulation of cells in the body may receive the Cas13d system.
But regardless of these current limitations, PAC-MAN’s effectiveness at targeting the virus responsible for one of the worst pandemics in the last few decades highlights a bright future for CRISPR-based vaccines in the future, which might completely revolutionize the vaccine industry forever. Till then, we’ll just have to continue social distancing and wearing masks to keep ourselves safe.
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On A More Personal Note:
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