How we can fight COVID-19 with CRISPR-Cas13
It’s about time we said “goodbye” to this virus.
The battle against COVID-19 has been a tough one.
As of today, there have been a total of over 95 million cases worldwide. Despite this, our battle with COVID-19 is nothing compared to the endless war being held in our oceans. Trillions die every day because of this war. What war is it, you ask? I’m talking about…
The war between Bacteria and Bacteriophages.
Ok, so what is a bacteriophage?
A bacteriophage is a type of virus that infects and replicates within bacteria (yes, bacteria can also be infected by viruses, not just humans). And don’t worry, bacteriophages can ONLY infect bacteria, so you’re safe 😎.
There are more bacteriophages than all organisms combined! In fact, about 40% of bacteria in the ocean die every single day just because of bacteriophages 🤯.
So why does this matter?
Well, you see, bacteria have had to deal with this problem for millions of years, and in the process, have developed a system to combat them. It’s called…
The CRISPR System
(pronounced “crisp-er”)
CRISPR, or Clustered Regularly Interspaced Palindromic Repeats, is essentially a bacterial immune system. You can think of it as a “library of viral mugshots”. Basically, after a bacterium manages to survive a viral infection, it keeps a copy of the virus’s genetic information (like an ID for the virus). This genetic information is stored within the CRISPR region of the bacteria’s genome. So, next time, if the same virus were to enter into the bacterium again, the bacterium would immediately be able to recognize it and cut it in half.
Scientists found that the “locate and cut” mechanism was performed by the Cas9 nuclease. Scientists also found that the Cas9 nuclease can be programmed to cut certain DNA strands with the help of a guide RNA. This now meant that scientists could make a cut virtually anywhere in the genome with the help of the CRISPR-Cas9 system ✂️🧬.
As great as this 2012 discovery was, it was only scratching the surface. Over the years, scientists have found (and even modified) many Cas proteins. For the purpose of this article, I’ll be focusing on…
CRISPR-Cas13
CRISPR-Cas13, like it’s cousin, Cas9, does the very same thing. Except, instead of cutting DNA, Cas13 cuts RNA.
Side Note: Why would there be a Cas protein that cuts RNA?
Because bacteriophages come in all sorts of various forms. Some bacteriophages carry DNA and others carry RNA. In order to defend themselves from all sorts of phages, bacteria had to develop Cas proteins that could target both DNA and RNA (💡 SARS-CoV-2 is an RNA virus).
Wait a second, if bacteria use CRISPR to defend against their viruses, what’s stopping us from doing the same thing against our viruses?
What if we used CRISPR-Cas13 to fight against COVID-19?
That’s exactly what many scientists were also thinking after the COVID-19 pandemic struck. In fact, scientists at Stanford University have already developed a system that uses CRISPR-Cas13 as an antiviral for COVID-19. They call it…
PAC-MAN. ᗧ - - ᗣ
Or (Prophylactic Antiviral CRISPR in huMAN cells).
As amazing as that acronym is, what’s more amazing is what it does and how it works.
Using CRISPR-Cas13’s ability to cut RNA, Dr. Stanley Qi and his team at Stanford University thought about how they could use it against COVID-19. If you didn’t catch it yet, COVID-19 is caused by an RNA virus. Because of this, CRISPR-Cas9 would be ineffective, as it is only able to cut DNA.
Cas13 works along the very same lines as Cas9 does. You can program it with a piece of RNA (called crRNA in this case) and Cas13 will search throughout the cell for a matching RNA sequence. If it finds that sequence, it cuts it in half. So, all the scientists need to do is give Cas13 a crRNA that targets specific sequences found in SARS-CoV-2’s genome 🎯.
This system is in contrast to modern-day antiviral treatments → (usually nucleoside analogues, viral entry inhibitors, or enzymatic inhibitors).
“Two of the more highly conserved regions contain the RNA-dependent RNA polymerase (RdRP) gene in the polypeptide ORF1ab region, which maintains the proliferation of all coronaviruses, and the nucleocapsid (N) gene at the 3′ end of the genome, which encodes the capsid protein for viral packaging.” (Qi et al. 2020)
The PAC-MAN system is especially different from vaccines. In the event of a SARS-CoV-2 mutation, PAC-MAN takes the slightly upper hand over vaccines. This is because the RNA sequences that PAC-MAN targets are highly conserved regions of the virus’s genome. In other words, PAC-MAN targets genes that are highly unlikely to mutate (due to the biochemistry of the virus). These genes are the RNA-dependent RNA polymerase (RdRP) and Nucleocapsid (N) genes. As well as that, PAC-MAN has been found to be up to 70–85% efficient.
If PAC-MAN is so great and all, then why isn’t it being used?
Aaaand this is where the catch comes in. 😬
There’s still a long way to go until we see PAC-MAN being used as a mainstream treatment. Unfortunately, we probably won’t see PAC-MAN be used during the COVID-19 pandemic. The technology is still in its early stages and it’ll take a few years to become mainstream.
“The biggest bottleneck is the lack of FDA–approved ways to deliver CRISPR components safely and effectively into the human respiratory tract and the lung” (Zhang 2020)
The biggest challenge that scientists face is the delivery of the PAC-MAN complex into lung cells 🚚📦. Some suggest using an engineered virus as a method of delivery (YES, a VIRUS — this method is actually more common than you may think). But, as you can imagine, inserting a viral particle into the body could cause an immune response and this would make the situation even worse.
Another challenge is off-target effects. The CRISPR-Cas system isn’t perfect and it can make a cut even if the crRNA doesn’t completely match the targeted RNA sequence (think back to how this could help the bacteria). Because of this, scientists are concerned about CRISPR-Cas13 making a cut in the wrong place (which could lead to even more severe problems).
A third challenge is the actual immunogenicity of the CRISPR-Cas13 components (Woah, that’s a big word). In other words, how will our immune system react to CRISPR-Cas13?
CRISPR-Cas13 is a foreign substance and could be deemed as harmful by the patient’s immune system. Also, recall where CRISPR-Cas13 even comes from… BACTERIA! Yes, not all bacteria are bad (you have trillions in your gut right now!), but it just so happens that the CRISPR-Cas13 system comes from bacteria that are pathogenic to humans 😷.
Here’s the TL;DR
Next steps for PAC-MAN include…
- Validating the efficiency and specificity of crRNAs for inhibiting infection of respiratory tract cells with live SARS-CoV-2 virus.
- Evaluating its immunogenicity (body’s immune response).
- PAC-MAN will need to be tested for its off-target effects.
- Validation in pre-clinical models (mice, ferrets, or lung organoid models).
- Validation through human clinical trials.
Does that mean that PAC-MAN is hopeless?
Of course not! (otherwise, I wouldn’t be writing this article 🤭).
Sure, you might not see it being used to combat COVID-19, but that’s assuming the pandemic will get over in the next few months (who knows how long it will actually take).
Besides that though, PAC-MAN’s scope of impact stretches far greater than just COVID-19. Remember those crRNAs that target certain parts of the SARS-CoV-2 genome? Well, it turns out that a cocktail of just 6 different crRNAs can target 91% of ALL Coronaviruses! This includes the infamous SARS coronavirus from 2003 and the MERS coronavirus from 2012.
But it doesn’t end there. The same scientists found that a cocktail of 22 different crRNAs could target ALL Coronaviruses known to exist in nature! This is pretty big news considering that we may have found a “penicillin-like” breakthrough for viruses.
One of the biggest challenges that PAC-MAN faces so far is the delivery of it into cells. But, scientists at Yale University think they’ve found a solution…
Introducing ABACAS. 🧮
OR (AntiBody And CAS)
scientists never fail to amuse me with their creative acronyms
Before I get into how it works, let’s do a quick recap of what problem ABACAS really solves.
How can we get PAC-MAN into Cells? (3 options)
- Introduce the PACMAN components in the form of DNA (DNA plasmid).
- Introduce the PACMAN components in the form of RNA (assuming that its mRNA).
- Introduce PACMAN as a ribonucleoprotein complex (shown to have a high editing efficiency + low off-target effects)
That’s good and all, but we still need to figure out a way to efficiently get these components into cells.
One idea is to use AAVs to deliver PAC-MAN. But…
→ AAVs don’t specifically target the infected cells (infected lung cells).
→ They will spread PACMAN into both healthy and infected cells.
→ This would lead to more off-target effects.
Therefore, we need a system that is both selective and efficient.
Hint: ABACAS holds the potential key to this solution. 🔑
SARS-CoV-2 has spike proteins on its surface. Our immune cells detect these spikes and create fitting antibodies that prevent the virus from getting into cells. Anyways, the key here is that antibodies can fuse with the spikes on SARS-CoV-2.
The writers of this research paper must’ve put the two together and thought, “what if we could fuse an antibody to the PAC-MAN complex?”
Well, that’s where the name ABACAS came from. ABACAS means AntiBody And CAS Fusion 💥.
In theory, with an antibody attached to Cas13, it wouldn’t need any help finding it’s way around. The ABACAS complex would just latch onto SARS-CoV-2 and travel with it inside the cell. Once inside the cell, SARS-CoV-2 would start to release its RNA genome. Just as this happens though, Cas13 would recognize the RNA and cut it in half immediately ✂️.
This makes the whole process:
- Selective: Cas13 only enters cells that are infected while ignoring healthy cells
- Efficient: Gets rid of any mistaken delivery into cells that are healthy. Only selects for cells that are infected.
The Drawbacks of ABACAS
Although ABACAS sounds like the solution to the delivery problem, there are some pretty big gaps.
Firstly, it’s one thing having a great idea, but it only becomes a reality once experimented. ABACAS is still a concept. It has yet to be tested in vitro (with cells that are in the lab/outside the body). There’s a chance it may work, and there’s also a chance it may fail. Each outcome would define the outlook of ABACAS.
Secondly, there hasn’t actually been an antibody identified as “the one” to use. This is obviously because, once again, ABACAS has not been tested or experimented with. The research paper only displayed good antibody candidates but didn’t define one as being “the best of them all”.
But wait, there’s more!
Congratulations on making it halfway through this article! Up until now, I’ve only discussed how CRISPR can be used as an antiviral treatment against COVID-19.
For both PAC-MAN and ABACAS, their stories are somewhat the same. They’re both fantastic ideas, but there’s still a long road ahead before we see them being used in our hospitals.
Fortunately, there’s another field where CRISPR is creating a lot of disruption: Rapid COVID-19 testing.
Currently, we use a method called RT-PCR to test for COVID-19. It’s based on the Polymerase Chain Reaction (PCR) which is used to amplify a piece of DNA. Since SARS-CoV-2 is an RNA virus, we need to convert the RNA into DNA, and then run PCR→ this is where Reverse Transcriptase (RT) comes in (RT-PCR).
There’s no need to understand the specifics of how RT-PCR works for the sake of this article. All you need to take away is that RT-PCR is SLOW. According to Public Health Ontario, it takes up to four days to receive test results.
In order for us to have more control over the pandemic, experts say that we must test as many people as possible. To add to that, COVID-19 can be asymptomatic in some people. Because of this, it is even more imperative that we find a way to speed up the pace of COVID-19 testing 📈.
Introducing SHERLOCK. 🔎
OR (Specific High-Sensitivity Enzymatic Reporter UnLOCKing)
I love these acronyms.
SHERLOCK works in a simple two-step process.
- Amplifies the RNA in the sample (creates thousands of copies of the RNA).
- Uses CRISPR-Cas13 to detect any viral RNA within the sample.
The amplification of the viral RNA happens in a process called Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA), a process very similar to RT-PCR.
Once the RNA has been amplified, Cas13 can start doing its job: searching for the viral RNA. Like we already know, if Cas13 finds any viral RNA, it will cut it in half.
But how will we know if Cas13 found any viral RNA?
This is where the beauty of the system comes in. Once Cas13 finds the RNA it’s looking for, it turns on this “psycho-mode” switch. When this switch is turned on, Cas13 will start to cut any RNA around it (even if it doesn’t match the target sequence).
Using this ability of Cas13, scientists add reporter RNA into the mixture. When reporter RNA gets cut (because of Cas13’s special ability), it glows fluorescent 💡. If scientists detect a fluorescent glow coming from the sample, they know that Cas13 has found viral RNA in the sample.
The best part about SHERLOCK is that the test only takes about 5 minutes to do! It’s also quite affordable and cheap. And if that wasn’t enough, you can do the test at home! To add on, SHERLOCK has already received FDA approval for their testing kit.
There are lots of other CRISPR-based diagnostics for COVID-19 as well. A notable example is Mammoth Bioscience’s DETECTR (DNA endonuclease targeted CRISPR trans reporter). It’s very similar to how SHERLOCK works. Except, instead of using Cas13, DETECTR uses Cas12.
Conclusion
CRISPR-based tools are truly an underdog in the field of viral treatment. These tools have promising outlooks and have the potential to fundamentally change how we treat viral disease. Although these treatments are fantastic in the lab, they still have a long way to go before we start seeing them used by our doctors.
On the flip side, CRISPR-based diagnostics are already changing the game. With exponentially faster results, cheaper costs, and simpler protocols, these tools may be the new standard of how we diagnose disease 🧪.
I think the moral of the story is this…
“If you see a problem in the world, go out and fix it…
It’s always worth a try”.
