Defending Against COVID-19 Using CRISPR CAS-13 and CAS-12
You are probably reading this article in the comfort of your own home. Why are you at home instead of at a cafe or work?
The answer is COVID-19. Our version of WW2.
I could go on and on about how it has affected our everyday lives, but that’s not what this is about.
You’ve heard all about the importance of social distancing and wearing masks, but today I’m looking at a way that can help diagnose COVID and future viruses but also hopefully be used as a therapeutic.
Although Pfizer and Moderna’s vaccine have shown incredible signs of success, there’s an opportunity that can work to cure a plethora of other diseases as well which is with CRISPR.
The problem with using these types of vaccines for future viruses as these are both mRNA vaccines.
This means that it will allow the body to create a harmless spike protein that fights against COVID. After the body creates this protein, the body gets rids of what told it how to make this protein.
It takes time for people to develop that mRNA sequence that is harmless to the body after the instructions are given.
We now know what we have to do to defeat the virus with these two vaccines, but this entire pandemic has taught us a thing or two about how we can address and fight against a variety of different viruses in the future.
As we’ve learned, one of the most important parts to eradicating any virus, especially an air-bound infectious disease like COVID, is the diagnosing procedure.
Diagnosing the Virus
We currently use a molecular diagnostic method called RT-PCR (reverse transcription polymer chain reaction) for COVID-19. This is a certain type of polymer chain reaction (PCR) that uses something called reverse transcription.
Polymer chain reactions are able to detect DNA, but COVID-19 is an RNA based virus. This is why we need the reverse transcription found in RT-PCR.
The reverse-transcription will amplify the RNA so that it can be expressed as DNA and be able to be detected by the PCR.
The DNA is then tested to find specific genes that they are looking for.
While it is simple to perform, an antigen swab test (the current method we use as a sample) is slow. According to Public Health Ontario, it takes up to four days to find out the results of the test.
Let’s look for something else that can speed up this process.
Using CRISPR instead of Polymer Chain Reaction (PCR) to Detect it
The method of using CRISPR to detect COVID-19 could use a variety of different methods.
The first we’ll look at is using something called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) created by Sherlock Biosciences.
SHERLOCK works in a two-step process that first amplifies the virus and then uses CRISPR to detect.
Let’s dive a little further into how this works, but first, a little about CRISPR in case you’re still new to the topic.
Clustered Regularly Interspaced Short Repeats (CRISPR) CAS-13
As seen in my last article linked here, CRISPR CAS-9 is almost like a scalpel that works to identify a strand of DNA in the body using something called the guide RNA and then cuts it out. When using CRISPR CAS-9 you are able to use choose the guide RNA and the strand of double-strand of DNA you would like to replace it with.
That’s CRISPR CAS-9, so what about CAS-13?
CRISPR CAS-9 focuses on changing a double strand of DNA, but CAS-9 rather focuses on a single strand of it. This is otherwise known as RNA.
CAS-13 uses a guide RNA. It looks for the strand of RNA inputted as it’s guide RNA and changes the bacteria to make it non-harmful.
How does it do this?
Well, unfortunately, no one knows. All that can be understood that in nature, certain bacterias use CAS-13 to stop the proliferation of them and prevent them from continuing to affect the bacteria they are in.
Back to Detection
SHERLOCK allows for not only a much quicker detection process (<90 minutes) but also makes it much easier for people to run the diagnostic procedure.
In the research paper, “A protocol for detection of COVID-19 using CRISPR diagnostics” by Feng Zhang, Omar O. Abudayyeh, Jonathan S. Gootenberg, several experiments were conducted to get the most optimal genes to use when looking for COVID.
SHERLOCK works in three steps
- Obtaining the sample
- Amplifying/Adjustments
- Inspecting
The two genes they chose to use to detect the virus are the S gene and Orf1ab gene.
The S gene, otherwise known as surface glycoprotein, in COVID-19 is responsible for the crown-like appearance the virus has, giving it its unique shape.
The Orf1ab gene is what is responsible for the encryption of two polyproteins called PP1ab and PP1a. These polyproteins help in allowing the virus to replicate itself.
They chose these genes as they are compared to other coronaviruses that are similar to COVID-19.
How do we know the genes are there?
Okay, so we know that when programmed correctly, the SHERLOCK system will be able to look for both the S gene and Orf1ab gene in cells, but how will we know they’re there?
The first thing the SHERLOCK system has to do is amplify the RNA in our bodies so that it can be more abundant to be detected and expressed as DNA.
The next thing we have to do is expressed the amplified DNA as RNA. You use a T7 transcription-polymerase to do this.
This can be done using LAMP, an isothermal amplification process. What’s special about this is that it can be done all at the same temperature.
The most important thing to do is to ensure that the CAS-13 protein has the correct guide RNAs.
When the gene is found by the CAS-13 protein after the guide RNA is added, it causes cleavage.
Because of this, SHERLOCK works. CAS-13 will be activated (fluorescent) when it finds a sequence of RNA it is looking for.
SHERLOCK works by allowing for the cleavage of it to produce fluorescence.
This is because the CAS-13 protein will have fluorescent protein on one side and a fluorescent mechanism on the other, if there is cleavage due to the detection of that RNA strand, COVID-19 is present.
RECAP of SHERLOCK method
- RNA is amplified and transcribed using RPA
- T7 transcription-polymerase is added to reverse-transcribe DNA back into RNA
- CAS-13 nucleases with a specified guide RNA (in COVID’s case they would have two different guide RNAs that are part of two different nucleases that are both found in COVID) are added
- CAS-13 will have cleavage if it senses the guide RNA. This cleavage causes it to be fluorescent
Viruses it’s already Detecting
One of the most amazing things about SHERLOCK is that it’s capable of detecting RNA diseases right now.
Zika is one of these viruses.
This proves that it can be used for any RNA virus or disease.
Because of SHERLOCK and the method of using it, the test can not only be accessible by people from their home but also exact results (down to single molecules) as a result of the amplification process.
End-Result
The two-step process for how SHERLOCK works can be broken down into more steps regarding the actual patient interaction.
- Obtaining a sample
- Warming up the SHERLOCK device
- Placing the sample in SHERLOCK mixture
- Looking for where the line is on the piece of paper
The craziest part about SHERLOCK is that you DON’T NEED A LAB TO DO THIS.
McGovern INSTITUTE describes the test result on the strip of paper as something very similar to a pregnancy test.
A Therapeutic Option to Nullify COVID-19 using SHERLOCK
We talked about detection, but what about an option to help eradicate COVID from the body.
This is different from the vaccines in the news right now by Moderna and Pfizer that are using mRNA to fight against COVID. These types of vaccines will provide information through using messenger RNA to our cells on how to build COVID-19 protein which will allow them to develop resistance to them.
So how is a therapeutic option to cure COVID-19 different?
The therapeutic option that could be used to cure COVID-19 is going to work through picking up on the virus and trying to break it down.
How will this work?
There are no set steps for this outline, but here is how this could possibly work:
- Input guide RNA into the CAS-13 nuclease.
- This guide RNA could be something like ORF1AB as it will stop the COVID virus from producing certain polyproteins essential for its survival.
- Use the system to cut out nucleotides that will render it useless
STOP COVID
Another very interesting venture with CRISPR is something called STOP COVID.
Instead of using the nuclease, CAS-13, it actually uses CAS-12. But, what’s special about the CAS-12 nuclease?
The CAS-12 nuclease cuts DNA just like the most commonly used nuclease, CAS-9.
The CAS-12 nuclease is different though because instead of making a double-stranded break in the DNA, it makes a single-stranded break.
This means after the amplification process, you don’t have to transcribe the DNA into RNA, making the process much easier. Nothing else in the LAMP of amplifying the RNA would have to change as well.
Research is what will allow us to continue making progress with technologies like these!
CRISPR is a very versatile tool that can be used to solve many different problems, including COVID!
Thanks for reading!
I’m Ali Haider, a 16-year-old high school student interested in a variety of different topics in the field of medicine.
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