Prime Editing Oversimplified
It has been 8 years since the first version of CRISPR-cas9 was discovered by Jennifer Doudna and her colleagues, and since then, gene-editing technology has evolved so much. Many variants of the CRISPR system have been developed, and new systems like Base Editing(BE), and now Prime Editing(PE), have been discovered. The basis of all these systems is that by changing the genetic lettering of a DNA strand, we can treat or cure a plethora of genetic diseases and disorders that were previously untreatable (Colour blindness, Breast cancer, e.t.c).
In late 2019, scientists David Liu, who developed the BE system, and Andrew Anzalone at the Broad Institute of MIT and Harvard created the prime editing system, taking inspiration from both BE and CRISPR. So what is this new Prime Editing system, and how does it stand against its predecessors? I’m going to attempt to explain PE and all the other baffling concepts of its sciences in such a way that my 8-year-old sister could understand.
First, let’s try to understand and visualize the DNA double helix. We can think of the DNA structure like a bridge, and let’s say that this bridge in particular has a faulty section.
In real life, people with single-gene disorders like sickle cell anemia have those conditions because their DNA has faulty lettering. This is called a point mutation, where a single out-of-place base pair in the DNA disrupts bodily functions. Imagine only one cable on a suspension bridge is out of place, but because of it the whole bridge and city(human body) that it is in face consequences. For example, in the case of sickle cell anemia, it is caused because of a single base letter substitution in the gene that is responsible for hemoglobin production in red blood cells. Lack of the protein hemoglobin that carries oxygen, leads to red blood cells that are in the shape of sickles, which cannot properly transport oxygen throughout the body. All of that just because of a single mismatch in genetic lettering!
We can solve these mutations using gene editors. Gene editors are capable of making their own point mutation changes and moving around base letters to where they are supposed to be, hence restoring order.
The people of the city want to fix the faulty bridge so they bring out the Prime editor bridge-repair system. Prime editors are made up of 3 main components: a cas9 nickase, a reverse transcriptase, and a pegRNA. “Woah! what are those?” you may ask, and I too was bewildered by all this foreign terminology at first. Simply they are a system of tools that work together to perform a job (the sophisticated job of editing DNA).
The cas9 enzyme is making a reappearance once again for the new PE system, but this time with a little twist. The cas9 nickase that is in PE is “crippled” unlike the usual cas9 enzyme, in the sense that it doesn’t make double-stranded cuts (DSC’s) like it normally does. I will go in-depth on why PE utilizes single-stranded cuts (SSC) later in the article, but for now, we can think about the cas9 nickase as the power saw of the system.
Next is the reverse transcriptase (RT), which has the job of synthesizing the desired DNA for insertion. Normally in the cell, the DNA is the nucleic acid that transcripts or produces its corresponding RNA. Whereas in the case of RT, it uses the given RNA sequence to produce its complementary DNA, hence its name. The RT in our bridge analogy is sort of like the 3D printer for bridge cables in the PE system.
Last but very much not least is the prime editor guide(peg) RNA. It is composed of a single guide(sg) RNA, a primer binding site(PBS), and the edited RNA sequence that the RT will use for transcription. Its main role is to guide the whole Prime Editor complex to the target DNA and to encode the RT with the desired RNA sequence for transcription. In terms of our bridge-building system, the pegRNA can be seen as the man lift crane and operator, this said operator also holds the blueprint for the reverse transcriptase.
These 3 components come together to make the *drumroll* PE:pegRNA complex…
and our bridge repair prime editor…
Now that we have identified the different parts of the system, we can look at how the “magic” happens. The PE process starts with the pegRNA finding the target DNA and giving the cas9 nickase the go-ahead to nip a single strand of the DNA. The crane drives towards the faulty bridge, and construction workers use the power saw to cut a section of the bridge.
After the cas9 nickase nicks the target DNA, a flap of the cut DNA forms. The primer binding site of the pegRNA docks to the DNA flap. The pegRNA then encodes the RT to transcript DNA complementary to the given RNA template. This RNA sequence in the pegRNA is pre-edited with the desired genetic lettering. The crane docks its platform to the half-severed section of the bridge, and the operator inputs the blueprint for the new bridge cables into the 3D printer.
Next, the RT reads the edited RNA sequence and transcripts the new desired DNA sequence, and the genetic lettering is attached straight on to the original DNA flap. The next part requires a little luck as the cell could either take back the original strand or accept the edited DNA. If successful, the target DNA incorporates the edited strand, and the original strand is cleaved by the natural cellular endonuclease. The 3D printer produces the bridge cables, and are mended on to the bridge. The old cables are removed.
“But wait, we have an issue!” you may exclaim, “we only repaired one strand of the DNA!”, and you are right. There is a mismatch in genetic coding between the edited and unedited strands of DNA. We can solve this issue by nicking the unedited strand, this causes the DNA to remake the nicked strand using the edited strand as a template, therefore matching the strands. It is difficult to explain this step using the bridge analogy, but for the purpose of the analogy, let’s say that bridge can heal itself. Finally, the construction workers damage the untouched side of the bridge, and this now sentient bridge uses the new cables as a blueprint to fix itself.
Using this process we can perform targeted insertions, deletions, and swap all 12 base combinations of the human cell; as advertised by David Liu and his fellow scientists to be a true “search and replace” system. Have I sold Prime editing to you yet? If not, let’s look at how it stacks up against its less flashy parents.
CRISPR-cas9 is much less complex than PE. The setup for CRISPR usually consists of a simple guide RNA, donor DNA, and our favourite cas9 enzyme, but this time with both of its nuclease domains intact. That just means the cas9 that is in CRISPR-cas9 can make double-stranded breaks(DSB).
DSB’s are in my opinion the biggest issue with the CRISPR system, as they cause a lot of problems for the affected DNA. The CRISPR system relies on the cell to heal itself after the cas9 enzyme cuts out the problematic DNA sequence, either via homology-directed repair(HDR) or non-homologous end-joining (NHEJ). The desired outcome for repair is usually via HDR, as it has the lowest amount of random INDELS, and uses the given donor DNA to fix itself, therefore incorporating the desired changes. If HDR fails, however, the cell uses NHEJ which is usually disastrous because the system is notorious for adding and deleting random bases, creating undesired mutations. The issue is that HDR has a significantly lower success rate compared to the cas9 cutting process. HDR efficiency hovers around 10%, whereas cas9 cleavage efficiency is relatively higher.
It is as if you were to cut off your arm to stop the spread of venom and then expect it to grow back without further implications. This is why PE utilizes single-stranded breaks(SSB), as it is a much more surgical approach and maintains the DNA’s structural integrity. PE also does not require any donor DNA to be inserted, due to the presence of the RT, which produces the desired DNA without the low percentage of HDR. Liu and his team conducted tests with PE and sickle cell anemia and Tay-Sachs disease and found the success rate to be between 35–55%, with the chance of random INDEL’s under 10%, PE definitely wins the race for precision and efficiency against CRISPR.
In regards to Base Editing, I think that BE takes the throne for the highest efficiency and fewest INDEL’s. David Liu, the scientist behind both BE and PE compared the 2 techniques and found that in situations where BE can be applied(C to T, T to C, A to G, or G to A base swaps), it is best to do so. I think that simplicity of the system is what makes BE so efficient. BE requires no strand cuts at all, but it is restricted to 4 base combination swaps. BE is like the layup shot of gene editing, it is simple and easy but gets the job done.
Where Prime Editing beats both of its predecessors is versatility and flexibility. PE is Base Editing and CRISPR in a combo package. PE can delete and insert long genetic sequences, like CRISPR, whilst also holding the ability to do ALL 12 point mutations(base combination swaps), which is even better than Base Editing. Prime Editing is the Jack-of-all-trades of gene editing.
Another factor that adds to PE’s flexibility is that it does not require the protospacer adjacent motif(PAM) to be in an optimal position for its complex to operate. The PAM is what the CRISPR system uses to identify which strands are targets and which aren’t, the PAM with the correct genetic coding must be present and in space a certain number of bases away for the CRISPR system to latch on to the DNA strand.
The PAM sequence is what restricts both the BE and CRISPR from targeting certain strands. You can use the logic that you wouldn’t park your car on top of parking lines, you have to park in the given parameters. PAM’s are like parking lines for guide RNA. Unlike its predecessors, PE does not require the PAM to be in a certain position, meaning it can hit all the target DNA regardless of PAM. This would increase the efficiency of therapies, as a larger group of cells can be altered.
Overall, I feel that PE wins the friendly competition for being the MVP gene editor. Yes, it still hasn’t seen as much testing and trial as CRISPR has, but PE is showing real potential.
“This is the beginning rather than the end, If CRISPR is like scissors, base editors are like a pencil. Then you can think of prime editors like a word processor, capable of precise search and replace… All will have roles.” ~ David Liu
Ok, so we’ve finally covered the major players in gene editing: CRISPR-cas9, Base Editing, and the rookie, Prime Editing. “All this new technology is cool, but why is it important?” you may ask, and the answer is clear. Genome editing is the next step in healthcare. David Liu has explained that Prime editing is capable of curing 89% of genetic human diseases. He is talking about numbers in the ten-thousands of genetic disorders; PE could save millions of lives around the world. Genome editing opens the scope to treat and cure diseases that have troubled humanity for centuries.
Gene editing could even be the solution to the pandemic we are amidst and future ones too. Imagine, 20 years later, we get hit with another pandemic, we would be equipped with the power of reliable gene editing. Gene editing would eliminate the concern of a pandemic even before it reaches the epidemic stage. Testing for disease would be revolutionized. Gene-editing based tools would be able to detect the virus in minutes, significantly reducing the spread. Humans could also be edited to be resistant to such a virus. The discovery of PE just brings us one step forward to that utopia.
There is also the side of gene-editing that focuses on enhancing the human species even further, the stuff you see in movies. We could create literal superhumans; people with enhanced strength, or enhanced eyesight. We could analyze certain genetic traits in animals that could be implemented in humans. I’m not talking on the calibre of wings, maybe something like increased breath capacity.
So what is stopping us from integrating genome editors into the real world? For one, we still don’t have gene-editors with a safe high percentage success rate. PE still has a chance of making off-target changes, which could cause an abundance of problems. Without consistent and reliable results, healthcare systems will continue to avoid gene editing as a viable option.
We also have to overcome the hurdle of social and ethical factors that play a role in the topic of genetically engineering humans. The first issues that come to mind are “Who gets it first?”, “What is the limit?”,”How would access be controlled between socioeconomic groups?”. People are also willing to create “designer” babies by editing the human embryo, but how would consent work? These are all issues that I hope we will solve in the foreseeable future.
The next steps for Prime Editing and the field of human bioengineering are making the safest and most efficient version of PE possible, solving the ethical issues that come with gene-editing, and finding a way to slowly integrate PE into the world of medicine while keeping equality in mind. Gene-editing is the future of healthcare, and PE has the potential to make a significant impact on society and medicine. We are now entering a new age; the thought of invincible humans is somewhat frightening, but gene-editing implemented the right way can boost the human to a higher peak, and I am excited for what the future beholds.
