It’s a prime time for Gene Editing! — PRIME EDITING
You probably came here because you wanted to learn about Prime Editing. Prime editing IS super cool and all, but before we talk about that, you need to know about DNA!
DNA: The molecule that makes you… YOU!
What do you, E. coli, and a sunflower all have in common? You all contain DNA! DNA, or Deoxyribonucleic Acid, is the instruction manual for our body. All of our traits, like eye colour and skin tone, are determined by our DNA.
DNA contains two strands that are bonded to each other in a twisted ladder or double-helix shape. Each strand contains its own sequence of nucleotides. A nucleotide is like “a letter in the dictionary of DNA”. In fact, nucleotides are actually labeled with letters! There are 4 different nucleotides: A, T, C, and G. Each nucleotide can only bond to another specific nucleotide. These are known as base pairs: A + T make a pair and C + G make a pair.
Short sequences of nucleotides are called genes, and each gene encodes for a specific protein. Proteins are the building blocks of our body, while genes are merely the instructions. When a gene is expressed, a specific protein is created. If the nucleotide sequence of that gene were to change, a different protein would be made. It’s like when you change the letters in a word, the meaning changes too! When the sequence of a gene changes, it’s known as a mutation. Mutations happen when a letter gets added or deleted from a gene sequence — this is known as an indel.
Mutations are harmless most of the time, but some can cause harmful proteins to be made. Mutations can happen at many times, like when a cell is replicating or when a cell is exposed to harmful radiation (like UV rays). Fortunately, there are many fail-safes put in place by our body to avoid mutations getting out of hand. One method is apoptosis, a process in which a cell exterminates itself. Unfortunately, sometimes mutations can cause the fail-safes themselves to fail, and this can result in atrocious things like cancer.
CRISPR-Cas9: The Breakthrough
The discovery of CRISPR-Cas9 truly was a breakthrough discovery and completely changed the playing field of gene editing. With CRISPR, scientists can target certain parts of DNA, cut it out, and add new parts in, all with surprisingly high accuracy. CRISPR opened so many doors that were previously closed.
Scientists could change pretty much any gene they wanted to. They can manipulate stuff like the eye or hair colour of an organism, all the way to treating genetic diseases like sickle cell anemia. CRISPR gave scientists a shot at actually dealing with cancer once and for all.
So, how does CRISPR work?
Let’s talk about bacteria for a minute…
CRISPR-Cas9 was originally discovered as a defense mechanism against viruses in the species of bacteria called Streptococcus pyogenes. Yes, you read that right, bacteria get infected by viruses too! In fact, around 40% of all the bacteria in the ocean get killed by bacterial viruses each day. This infamous family of viruses, formally known as bacteriophages, have been at battle with bacteria for millions of years. Phages infect bacterial cells by injecting their own genetic information into the cell. This viral DNA contains information to create new viruses. Essentially, the virus hijacks the cell and uses it as a factory to produce more viruses. These new viruses then quickly assemble, and tear the cell apart upon their exit.
In order to defend against these pesky viruses, bacteria created the CRISPR system. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is like a bacteria’s very own “library of viral mugshots”. Basically, when a bacteria realizes its getting infected by a virus, it sends out two enzymes called Cas1 and Cas2. These two enzymes locate a piece of viral DNA and shred it to pieces, therefore disabling the virus’ ability to replicate. Before they do this though, they keep a little bit of DNA for themselves, and store it in the “CRISPR section” of the bacterial genome. Now the bacterium has a copy of the viral DNA, so if the same virus were to attack again, it could recognize and destroy it immediately.
But bacteria don’t have eyes, so how can they “see” if a piece of DNA matches the viral DNA in the CRISPR section? This is where Cas9 comes in.
Cas9, or CRISPR-associated protein 9, is a type of nuclease. Nucleases are enzymes that can cut specific parts of DNA. Nucleases are completely natural and exist in human cells as well. In fact, nucleases are essential machinery for many different aspects of DNA. Cas9 also keeps a “template” of RNA with them. It’s this property of Cas9 that makes it so special… but more on this later.
Anyways, what role does Cas9 actually play in the bacterium?
The Cas9 nuclease is sent in and keeps a “template” of the viral DNA with it. Like I mentioned, this template is made out of RNA, not DNA. The Cas9 nuclease uses the RNA template as a “puzzle piece” that only fits together with another specific DNA “puzzle piece counterpart”. When the Cas9 nuclease finds a match, it immediately knows that it’s the same viral DNA as before, and so it proceeds to cut it.
A quick note about RNA if you’re not familiar…
RNA, or Ribonucleic Acid, is very similar to DNA, in the sense that it is made up of a sequence of nucleotides. Unlike DNA, RNA usually comes in one strand. Also, RNA has the letter “U” instead of “T”, and it still bonds to “A”. RNA is awesome because it can bond to unfolded pieces of DNA in a process called transcription. Hint: It’s THIS property of RNA that plays a big part in the CRISPR-Cas9 system.
Anyways, scientists discovered that they could program the RNA template to search for whatever sequence they wanted. When the sequence is found, the Cas9 nuclease would make a cut for them at that specific point. In other words, you can think of Cas9 as a pair of genetic scissors that make tiny cuts in DNA, and the RNA template as a genetic lock that only opens when it is presented with the right password. When you put two and two together, you get a pair of tiny genetic scissors that can be programmed to make a cut wherever you want to in the genome of an organism.
See where this is going? Scientists now had a tool that could search and replace specific genes! With this ability, scientists could actually cure lethal genetic diseases like cystic fibrosis! The best part about all this is that it could be done on a live organism too!
Ok, now time for how it works…
First, scientists engineer an RNA template for the Cas9 protein to use; this template is known as the guideRNA or gRNA for short. It basically tells the Cas9 what to look for. Next, scientists insert the gRNA into the Cas9 protein. Once this is done, the Cas9 protein goes to work and finds the specific gene sequence. It then makes a cut at the PAM site. The PAM (short for protospacer adjacent motif) is a region, around 3 base pairs long, that tells the Cas9 to “cut here”.
Normally, once the cut is made, the cell will immediately try and repair it by bonding together random base pairs. This is known as non-homologous end joining, and it can result in dangerous mutations in the sequence. To avoid this, scientists can send in donor DNA. The donor DNA would be very similar to the DNA just cut out, except with one small edit made. The cell notices this high similarity and can opt to use the donor DNA instead of random base pairs. This new approach is known as homology directed repair, and is a much safer option than non-homologous end joining.
There are still a few caveats though…
Non-homologous end joining can still occur even with the donor DNA nearby. In fact, only some cells in the body can use homology directed repair. Therefore, the scope of what CRISPR-Cas9 can edit is greatly reduced. Also, even if a cell can use homology directed repair, it is still up to chance whether it actually will or not. Scientists simply hope that the donor DNA will do its job!
The CRISPR-Cas9 system also has the potential to do more harm than good. This is because the Cas9 protein doesn’t always make perfect cuts, and these are known as off-target edits. Sometimes, Cas9 will find a DNA sequence that looks “very similar” to the sequence that the guideRNA outlines. There may be a few base pairs that are different, but the Cas9 protein will excuse these and make a cut anyway. Although this doesn’t occur very often, there is still a chance. A cut in the wrong place can cause undesired mutations, and this can lead to even more problems. One of the main reasons this occurs is because Cas9 makes double-stranded breaks in the DNA, meaning that it cuts both strands of DNA.
Fortunately, Prime Editing solves this problem!
Prime Editing: CRISPR 2.0
Prime editing was developed by researchers at the Broward Institute of Harvard and MIT in October 2019. This new technology is a modified version of the previous CRISPR-Cas9 mechanism, and shares many similarities.
Prime editing has three main components:
- Cas9 Nickase
- pegRNA
- Reverse Transcriptase
1. Cas9 Nickase
The Cas9 nickase is essentially a modified version of the regular Cas9 enzyme. This version of the enzyme only has one “genetic scissor” rather than two. This modification allows it to make only single-stranded cuts. Therefore, this enzyme simply nicks the DNA, hence the name “nickase”. The Cas9 nickase solved two of the biggest problems that the original Cas9 had — off-target editing and NHEJ. With the Cas9 nickase, off-target edits are highly less likely to occur, since Cas9 is looking to make a cut on only one specific strand that matches. Also, the problem of non-homologous end joining is solved, as it only occurs during double-stranded breaks and not single-stranded breaks.
2. pegRNA
pegRNA, or Prime Edited guide RNA, is a modified version of the original CRISPR guide RNA. pegRNA is much larger than the original gRNA in size, and this gives it the ability to “stretch further”. Because of this ability, the pegRNA is able to latch onto both strands of unzipped DNA! This gives it way more stability than the original CRISPR system, which only latched onto one strand of DNA.
3. Reverse Transcriptase
Chances are that you’re not already familiar with the Reverse Transcriptase enzyme, so let me give you a little lesson on viruses…
Viruses come in both DNA and RNA variants. DNA viruses have no problem in hijacking the host cell as all the genetic information in the cell is DNA and therefore it can integrate without worry. RNA viruses don’t have it this way. You see, you can’t just fit RNA into a sequence of DNA, they both have different chemical backbones (deoxyribose vs. ribose).
To solve this, RNA viruses use a special enzyme called Reverse Transcriptase. Remember that process called transcription? Well, this enzyme does the exact opposite! This special process, known as Reverse Transcription, converts RNA into DNA! Therefore, the viruses use this enzyme to convert their RNA into DNA, so it can be integrated with the host cell’s DNA.
This SAME enzyme is used in prime editing, and it is a key part of the entire process.
So, how does Prime Editing work?
The beginning of the process is quite similar to the original CRISPR-Cas9 method. Scientists make a specifically engineered pegRNA that will serve as a guide for the Cas9 nickase. Scientists will then need to attach the pegRNA onto a “Cas9 nickase + reverse transcriptase complex” in order to fully form the Prime Editing Complex.
The PE complex navigates to the specified target location in the genome and unravels the DNA. Then, it makes a single-stranded break on the PAM site. The pegRNA then latches onto both strands of DNA. On one strand, we have our target sequence, and on the other, we have it’s inverse sequence. The strand containing our target is fittingly known as the target strand, while the other strand is, you guessed it, known as the non-target strand.
Great! So now we’ve cut our DNA, but how do we make an edit 🤔? In the previous CRISPR method, donor DNA had to be added to the sample, and from there, it was simply up to chance that everything worked properly. With prime editing, all of that changed; here’s where prime editing really shines🌠.
The pegRNA is divided into two main components. One part actually bonds to the target strand itself. The other part acts as a reverse transcriptase template. This template is very similar to the piece of DNA we’re replacing, except for one small change in the sequence. Therefore, when the pegRNA bonds to the target strand of DNA, the reverse transcriptase immediately gets to work. The reverse transcriptase uses the piece of DNA bonded to the pegRNA as primer DNA. Primer DNA is essentially like a spark plug, but for reverse transcriptase. The reverse transcriptase, now primed, uses the template (located on the pegRNA itself) to construct DNA. This newly constructed DNA is automatically appended to the end of the primer DNA.
In essence, prime editing solved one of CRISPR’s biggest downfalls! With CRISPR, scientists would need to add donor DNA into the actual sample, and would also have to hope that it even worked. With prime editing, this could all be done by just inserting one complex!
So are we done? Well, not really… 😬.
Because we cut a strand of DNA into two, there are now two “flaps” of DNA that are located on the same strand (the target strand). One flap contains our edit, while the other flap contains regular genomic DNA. These two flaps would constantly be competing for a spot in the genome. Fortunately, we can send in another nuclease to cut this unedited flap off.
But now we have yet another problem🤦♂️.
Our target strand, which contains our edited piece of DNA, will have a mismatched base pair in our non-target strand of DNA. The cell will recognize this discrepancy, and will try to fix it immediately. The cell will either replace the base pair on the non-target strand (which is good), or replace the base pair on the target strand (what we really don’t want). One way scientists can trick the cell into replacing the non-target strand base pair is by creating a small nick on the non-target strand. The cell will see this nick and will deem that strand as unhealthy. Due to this, it is more likely that the cell will replace the base pair on that strand as it is already damaged. Although this outcome is highly likely, there still isn’t a concrete guarantee that the cell will actually do this, it’s still up to chance.
So to sum it all up, here’s a quick recap of the prime editing process:
The three main components
Cas9 Nickase (The “scissors”)
pegRNA (The “guide + template”)
Reverse Transcriptase (The “RNA → DNA converter”)
The process
- PE Complex navigates to the target site and makes a cut at the PAM
- pegRNA then binds to the target strand
- Reverse Transcriptase starts creating DNA out of the pegRNA template; Uses the binded target strand DNA as a primer
- There are now two DNA flaps: Edited and Unedited flap. The unedited flap is cut off with the help of another nuclease
- There is now a discrepancy between edited strand nucleotide and unedited strand nucleotide. To solve this, Cas9 nickase makes a cut on the unedited strand to trick the cell into replacing the nucleotide on that strand.
And there you have it! A permanent edit in the genome of an organism!! 🤯
The Bright Future for Prime Editing
Prime editing has made gene editing more safer and easier than ever before. With prime editing, scientists are able to make cuts in DNA that are more precise than CRISPR-Cas9 ever could. This unlocks the potential for doctors to cure a wider range of diseases, with more certainty that the treatment will be successful.
Although prime editing is an absolutely awesome invention, it may not fully take over CRISPR-Cas9 just yet. Prime editing was developed less than a year ago, and CRISPR is already being used quite often. What prime editing has shown us is that there is hope in improving CRISPR. All it takes is a little creativity and thinking out of the box. Prime editing may not be the technology to be used in the future, but it certainly has played a big role in paving the path forward.
Key Takeaways
Woah! I understand that that was A LOT of information to digest — trust me, I’m the one who did all the research😅. To help you remember what you just read, here’s a very quick recap of Prime Editing:
- It’s a modified version of the CRISPR-Cas9 system
- Is like a search-and-delete function but for DNA
- Has way more control and accuracy over Cas9 system
- Higher efficiency/success rate
- Able to treat many more types of genetic diseases
- Much easier to implement/less interventions needed
- Still not perfect, but will play a big part in shaping the future
Why Prime Editing and CRISPR aren’t ready… yet.
A lot of aspects of CRISPR and Prime Editing are still up to chance. Despite this, researchers are looking into how to make these technologies as efficient as possible, meaning, “how much can we increase the likelihood of this working”. In actuality, these technologies are getting more efficient as time goes on. We’re not really that far off from a future where we have genetic editing as common treatments in hospitals. We’re not far off from a future where gene editing is accessible to the public.
But..
At the end of the day, there still isn’t a 100% guarantee that they’ll work every time. In fact, there may never actually be a 100% guarantee.
But that isn’t necessarily a barrier — it’s more a red herring. Many of the current treatments in our hospitals aren’t 100% guarantees either, yet patients still get cured everyday. In fact, a lot of the things we do in life aren’t 100% guaranteed. Life is all about taking chances, and when we do succeed, there’s always a wonderful surprise waiting for us at the end of the tunnel. The first astronauts were taking a huge chance when we sent them to the moon. The Wright Brothers took a huge leap of faith when they tried to make an aeroplane. What if they had never taken the chance? What kind of a world would we live in?
Science is all about taking chances. It’s about stepping into the dark realm of the unknown with only a matchstick. It’s thoughts like these that can make us feel hopeless at times, but it’s these same thoughts that fuel our curiosity. Knowing that there’s so much left unexplored, so much to discover, and so much to learn is a blessing in disguise. How boring would life be if there was nothing new to do?
So I encourage you to go out there. To be optimistic. To be brave and To push through even when it gets hard.
Even if there isn’t a perfect 100% chance that it’ll work, go for it anyway. Know that if you quit, there’s a 100% chance that it won’t ever work.
