Prime Editing: The Future of Gene Editing?

Aditya Dewan
14 min readJul 6, 2020

A new way to edit genes!

B y now, you’ve probably heard of CRISPR: the revolutionary tool that, for the first time, allowed us to cut and edit genes, much like a pair of scissors (even if you haven’t, don’t worry, we’re going to walk you through everything step by step). For about a decade now, CRISPR was the main way that people would edit genes.

However, because of some recent developments, it appears that CRISPR may not be the ideal tool to use for gene editing. Instead, we’re going to take a look at a recently discovered (and much safer) alternative to CRISPR: Prime editing.

What is CRISPR and Gene Editing?

Before we explain Prime editing (and how it could potentially unlock a new door for our species), you should first understand what CRISPR and gene editing are and how they work. Every living organism consists of cells, and these cells are the building blocks of all living things. In fact, there are an estimated 30 TRILLION cells in your body right now, all of them multiplying and working very hard to keep you alive.

Figure 1: A plant cell under a microscope.

Think of your body as a factory, and your cells as the factory workers. The factory workers drive machines that do various things, such as pump blood, digest food, get rid of waste, etc. Inside the center of these cells is a “chromosome”, which has of sections of genes inside it. These sections consist of DNA: the blueprint for life as we know it. DNA is like a blueprint, telling your body how to do different tasks and what proteins to make.

For example, one part of your DNA might be responsible for your eye colour, while another might determine your height, anything ranging from the shape of your ears to how many fingers you have. Under a microscope, DNA almost looks like something from science fiction, having a double-helix structure with a middle section composed of 4 different bases: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). A always pairs with T, while C always pairs with G. There are three main functions of DNA:

1. To form proteins and RNA 🤔(kind of like ½ of DNA, and carries protein blueprints from DNA for the rest of the cell to use)

2. Enabling mutations 🧬(mutations are basically just random DNA changes that happen from time to time)

3. Exchanging genetic material 🔄

For DNA to make these proteins that run our bodies, it needs to be “read” by a special type of RNA called messenger RNA, or mRNA for short. Imagine an old-fashioned computer with a CD slot. Think of DNA being like a CD, and the RNA being the computer that reads the disk and shows its content on the screen (or, the parts of the cell that need to make the proteins).

The RNA reads the bases in groups of 3s, and pairs each base with its corresponding one. Every one of these groups is responsible for a different protein building block, meaning that even a change in a single letter can result in an incorrect or different protein.

It’s quite amazing how something as small as DNA can have a huge impact on our bodies!

Ever since humans have discovered these instructions for life, humans have been trying to tamper with them. After all, if we can change the instructions making an organism, then we can alter the organism itself in amazing ways that help us! Longer food shelf life, glow in the dark fish, and mosquitoes that don’t spread malaria are within reach (and have all been accomplished) by messing around with DNA. This is known as gene editing, changing the genes (sections of DNA) that make up an organism.

Figure 2: The instructions for life, DNA.

Gene editing has one main goal: to get better traits from organisms that might help us accomplish a certain task. For example, let’s look at one of the major problems in agriculture just a couple years ago: insects. Often, bugs and other insects feast on crops, chewing their leaves, boring within the plant roots, stems, and leaves, and spreading diseases within plants that may cause an entire yield to perish. Since we collectively hate insects destroying our food, we decided to spray over 5.6 billion pounds of pesticides per year on our fields, killing the insects as well as our budget.

Figure 3: How BT Crops work.

Instead of using pesticides, which harm animals and the ecosystem around them, we can change crops to make their own pesticides. Cool, isn’t it?A crop that can make and use its own pesticide means no more runoff and ecosystem damage, no more expensive pesticides, and no more insects chewing on our yields. These crops (known as BT crops since they basically “borrow” a gene from a type of bacteria with the same name) are a perfect example of how genetic engineering can drastically improve our way of life.

Before CRISPR, one of the only ways to “engineer” organisms were to bombard them with radiation. Radiation weakens and breaks up DNA, and the idea was to use this on organisms to get a useful mutation by pure chance which, quite obviously, was not a good way to engineer organisms, nor was it safe for the scientists operating these machines. This was until we discovered CRISPR, a revolutionary tool that transformed genetic engineering forever.

CRISPR is a sort of DNA archive present in bacteria as a defense system against viruses, which viciously hunt bacteria. When a virus attacks a bacterium, it injects its own DNA inside the bacterium to kill it, and if the bacteria somehow survives this attack, it saves the injected DNA into an “archive” known as CRISPR. While it does sound like a breakfast cereal, CRISPR literally had the power to change our society as we knew it.

Moving on, when another virus attack occurs in the bacterium, the bacteria looks at the DNA and tries to see if it’s the same one that was injected inside it before. A special protein called CAS9 extracts that piece of DNA from the CRISPR archive and attempts to match it with any piece of DNA found inside the bacteria.

If it finds a match, then the virus DNA is cut off, rendering it useless. Take that viruses!

The genetic revolution began when scientists found that CRISPR and CAS9 are programmable and can be used to cut and edit genes in living organisms. We had found a hand to operate the pair of genetic scissors.

How does CRISPR work?

Figure 4: CRISPR uses Guide RNA (essentially DNA cut in half) and scans the gene’s DNA for a match. When one is found, it uses enzymes at the ends to cut off the DNA.

Now that we’ve gone over gene editing and CRISPR, let’s take a closer look at a simple version of how they work. CRISPR uses a protein called Cas9, and in the usual CRISPR/Cas9 system, a special version of our friend RNA (called guide RNA/gRNA) finds the area of the DNA that we want to snip with our scissors.

The gRNA is made up of two smaller RNA parts (as if that wasn’t enough RNA for one day): tracrRNA, and crRNA. While it sounds a little complicated, these parts are responsible for “locking in” the Cas9 protein to the DNA, and recognizing the various parts of it.

Then, we have something called PAM, which stands for protospacer adjacent motif (quite a mouthful!). In simpler terms, PAM is like the blades of the scissor, found on either end of the Cas9 protein and is responsible for recognizing and cutting the DNA ends. Without PAM, Cas9 wouldn’t be able to cut and edit genes.

Lastly, the cell needs to be provided with “donor DNA” that contains the desired changes and edits, using it as a “template” of sorts. Donor DNA is what Cas9 will use to find the part of DNA that it needs to match and cut off. Phew! That’s (almost) all the chemical jargon we’re going to need to explain Prime editing!

The Problem With CRISPR

Of course, for every yang, there is a yin. While CRISPR is insanely effective, there is one MAJOR problem with it that can potentially shut down human applications of CRISPR. Sometimes, the CRISPR protein makes double-strand breaks within DNA, which have recently been discovered to be extremely dangerous to the cell that the break occurs in.

Double strand breaks (or DSBs for short) are where both strands of DNA are cut, and this is harmful for a lot of reasons. First of all, DSBs activate the cell’s response system to try and “fix” the break. Unfortunately, this response system is extremely error prone and can cause even further damage to the host cell.

On top of that, double strand breaks are mutagenic, meaning that they can cause or increase the chances of a mutation. Mutations are basically variations in DNA that happen by pure chance, and while some mutations may be harmless, others may be extremely dangerous and cause genetic disorders, even leading to cancer in some cases. In addition to this, double strand breaks can also cause translocations in DNA, which is just a fancy way of saying that it can move itself to other areas where it isn’t supposed to be. This can cause even more problems depending on where the DNA has been moved to. These double-stranded breaks (or DSBs for short) also lack precise edits, since they can result in a complex mix of DNA and other products and sacrifice accuracy.

Figure 5: The negative effects of Double Strand Breaks (DSBs).

As if those weren’t enough drawbacks, DSBs can also result in the activation of p53. P53 is kind of like a police officer, making sure that the cell doesn’t duplicate itself uncontrollably and cause a tumor. The problem is that DSBs activate this protein even if the changes are intended, so the genetic police officer then takes action to prevent the cell from multiplying. This can result in a range of horrid effects, from death of the cell (in complicated terms, this is called apoptosis) to prevent it from multiplying, or senescence, where the cell loses its power of division and growth. The fact that CRISPR’s causes unsafe DSBs is a much larger issue than most people realize. If genetic editing is to be used in real world therapy, cure diseases, and help us to live a better quality of life, it must be safe to use, which simply doesn’t work with the version of CRISPR we use today. So, what’s the solution to this dilemma?

While there have been quite a lot of potential solutions to this problem, none of them are both safe and effective. If we want to make our genetic scissors safe, it needs to either make single strand breaks (SSBs) or no breaks at all. In other words, we need to upgrade our scissors to safety scissors. With single strand breaks, we can use Cas9 nickases (a version of Cas9 that causes one strand breaks) that only cut one strand. However, while these SSBs are safe, they are incredibly ineffective. SSBs can be quickly repaired by the cell, meaning that there isn’t enough time available for the edit to work.

Figure 6: Single Strand Breaks (SSBs) are safer than DSBs, but aren’t as effective.

Then, we have a way of genetic editing that doesn’t cause any breaks. To perform genetic editing without breaks, we can use a dead form of Cas9 that is can’t cause breaks but can change each individual base/letter. While this does “work”, it is extremely limited in the types of changes that it can perform since it can’t change large groups of DNA quickly. It’s also prone to off-target changes, and sometimes changes bases located in the wrong spot.

So, what do we know? Well, we know that our potential solution needs to be both safe and effective. To be safe, it needs to have either single strand breaks or no breaks at all. And to be effective, it needs to be able to perform 3 different actions: substitution (substitute bases with other bases) deletion (deleting bases), and insertion (being able to insert DNA). Our solution should also be able to perform a combination of these techniques.

So, how do we fix this?

Prime Editing

If CRISPR was like a map, Prime editing would be like a GPS: A “2.0” version of CRISPR.

Prime Editing is gene-editing tool that DOES NOT need DSBs OR donor DNA. Pretty amazing, right? The goal of prime editing is to make gene editing efficient and safe, because prime editing inserts DNA edits that researchers want to make into the DNA itself. This is unlike CRISPR, which uses the cell’s repair system to make the changes.

Figure 7: A basic diagram showing how Prime editing works.

This DRASTICALLY reduces the amount of accidental changes made to the genes (oops, we accidentally rearranged the cell), since the edits only take place in one strand.

By using a weaker form of Cas9 that’s given instructions with pegRNA (another form of RNA responsible for making the edit that the researcher wants), we can make both safe and fast edits. In fact, scientists from the journal Nature explain how they conducted upwards of 175 edits in human cells with excellent accuracy, including all 12 types of point mutation.

For reference, a point mutation is a type of mutation in DNA/RNA in where an individual base is changed in some way. This level of accuracy and capability gives researchers significantly better control over what edits are made.

Adding on, Prime editing doesn’t immediately activate the cell’s repair system, preventing premature cell-death and senescence. Whereas CRISPR requires researcher intervention for almost any action, Prime editing can accomplish all these functions without additional modification.

As if those weren’t enough benefits, this means that we can also repair harmful genetic mutations for various genetic diseases. Take Sickle Cell Anemia for example. By changing the genetic makeup of some stem cells in the bone marrow, scientists might be able to cure this disease!

In fact, the scientific director at the Innovative Genomics Institute believes that Prime editing can potentially be the way that mutations that cause diseases are cured, though he also believes that it is too soon to be sure, which is one of the primary issues with Prime editing.

However, just because Prime editing has REVOLUTIONARY benefits doesn’t mean that there aren’t any drawbacks. First, the prime editor is pretty large, and it won’t fit into the viruses that researchers have traditionally used to edit genes. And, we still don’t know whether Prime editing can edit large sequences of DNA both effectively and accurately, since not much testing has been done.

Second, Prime editing still results in unintended changes (still lower than CRISPR), meaning that it can still change the gene sequence so that it might make new or different proteins.While this may seem like nitpicking, we need to make sure that almost no problems are caused if we edit genes in humans. It’s something that we need to understand so that the safety of our edits isn’t based on pure chance.

GM watch, an independent organisation that provides news and information on GMO crops (crops that have been genetically modified) believes that the hype and excitement behind prime editing is “premature”, and doubts “whether it will even be used to develop GM crops and foods.”

Figure 9: Gene editing is widely used in crops for a variety of reasons, ranging from increasing shelf life to generating pesticides.

This opposition comes from similar claims of accuracy, safety, and precision that have been made by a variety of different institutions over the years, and, almost always, some problems show up and render the technique useless. organization rightfully believes that it might be a matter of time before more problems are discovered with Prime editing as well.

David Liu, consultant and co-founder of Prime Medicine (an organization dedicated to bringing Prime editing to humans), brings attention to the fact that while Prime editing did have fewer errors than CRISPR, it still had errors, though this is sometimes downplayed. Dr. Antinou agreed with this, stating:

“Prime Editing is an elegant but complex technique involving multiple components and molecular manipulations. The authors [of the paper] emphasise that it needs a lot more work before it is ready for clinical use. There are still too many bugs and unknowns.”

The bottom line here is that we simply don’t know enough about prime editing to replace CRISPR with it. Prime editing is a relatively new gene editing technique, and only became somewhat known in 2019. As a result, there hasn’t been enough research and development to truly identify if prime editing is safe. A little over 10 years ago, we didn’t know the severe negative impacts CRISPR could have.

While it is excellent that Prime editing may be a viable alternative, it’s too early to tell whether Prime editing will end up as a revolutionary therapy or an ineffective and unsafe treatment.

So, what might the future of Prime editing hold for us?

The Future of Prime Editing

So far, we know that while Prime editing is an amazing tool, it can also still have negative impacts. What does the future hold for our new gene editing scissors?

Figure 10: How a potential gene therapy might function.

The goal of any gene editing tool is to achieve a viable gene therapy using it, which would be able to cure thousands of diseases and genetic disorders, allow us to live longer, and maybe even help us adapt to our environment in a better way. With a tool like gene editing, there’s no limit to what we can accomplish. Once prime editing has been properly tested and clinical trials have been run, institutions such as Prime Medicine could bring Prime editing to humans.

David Liu and Andrew Anzalone (a researcher who oversaw the Prime editing project) estimate that at least 89% of known disease-causing genetic mutations can be theoretically corrected by prime editing.

89 percent!

Think of the sheer amount of lives we could save if this was perfected, the amount of people that we could save from a premature death! But prime editing is only the beginning of gene editing. Alexis Komor (who worked on base editing with Liu) states:

“Everyone needs to start working on this: how do we modify both strands?”

Think about it. While editing one strand at a time effectively is the current goal, it is by no means a limitation. What if we could find a way to edit both strands simultaneously? We could reduce the amount of time gene editing would take and also increase its effectiveness!

The sky’s the limit when it comes to the future of prime editing and genetic engineering.

Conclusion

Gene editing is, at its core, an insanely complex topic, and we have only scratched the tip of the iceberg when it comes to it. There’s still so much that we don’t know about editing genes, but we’re also working on finding out new and safer ways to edit them into organisms. Whether Prime editing is successful is still up for debate, but what we can be certain of is that humans will eventually find a way to achieve the impossible. Just over a century ago, we thought that aircrafts were impossible and that humans could never fly. In today’s age, flying has become one of the most popular ways to travel and transport goods. What we think is impossible today, may just become our reality in the future, and it could just be a matter of time before genetic engineering becomes a part of our daily lives.

Hope you enjoyed this article!

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Aditya Dewan

Building companies. Machine Learning Specialist @Actionable.co. Philosophy x Tech.