It’s PRIME TIME! — An In-Depth Look at Prime Editing

Introducing to the stage…PRIME EDITING! A more precise, search-and-replace gene editing technique.

Eliza Aguhar
Nov 3 · 8 min read

If you’re writing an essay and you’ve been accidentally spelling someone’s name wrong the entire time, you aren’t going to sit there and find every instance of that person’s name to correct it. You’re going to open the ‘Find and Replace’ shortcut, type in what you are looking for and what you want to replace it with. Once you hit ‘Enter,’ mistake would instantly be corrected and now, you won’t look like an idiot.

Now, imagine if you could do that with your DNA.

The new prime editing technique allows for a true search-and-replace ability when editing genetic code. My nerd level when it comes to gene editing: 1000000. (Photo from ABC).

In the world of molecular biology, the discovery of CRISPR-Cas9 as a gene editing method was revolutionary. Recently, the discovery of the new “prime-editing” technique by David Liu and Andrew V. Anzalone with the Liu Group means that gene editing is now more precise than it ever was before.

If CRISPR-Cas9 was a Magic Eraser, and erased any segment of DNA you wanted…

“You can think of prime editors to be like word processors, capable of searching for precise DNA sequences and replacing them.” — David Liu

Before we dig deep into prime editing, here is a quick refresher of some key things you should know:

  1. DNA has two strands and is shaped like a twisted ladder or a double helix
  2. Each stand is coded with a four-letter alphabet: A, T, C, and G. These letters form complementary base-pairs: A only bonds with T, C only bonds with G.
  3. One of RNA’s function is copying the DNA’s code. It bonds to an “unzipped” strand of DNA by creating a sequence of the code’s complementary base pairs. In RNA, A bonds with U instead of T.
  4. Mutations occur when the order of the genetic code is changed. Gene editing is essentially artificially inducing and specifying a mutation.
  5. CRISPR-Cas9 is a gene editing technique that allows you to edit a specific segment of DNA.

Speed round: What is CRISPR-Cas9?

For those of you who want a brief explanation of gene editing with the CRISPR-Cas9 method, this section is for you. There are two main components to editing using the CRISPR-Cas9 process: the guide RNA and the Cas9 protein.

The guide RNA (or gRNA) is a special type of RNA that can locate any part of the DNA. The coolest part of gRNA is that it can be programmed, meaning that if the scientists know what a DNA sequence looks like, they can program the gRNA to find that segment of DNA (also known as the target DNA) in any cell.

Once the scientists program the gRNA, they then insert it into an endonuclease called the Cas-9 protein. Endonuclease and Cas-9 seem like complicated words but just think of them as fancy, genetic scissors. The gRNA guides the Cas-9 protein to the target DNA, hence the name. Once the DNA is located, the Cas-9 protein cuts the target DNA at a section called the protospacer adjacent motif (or PAM). That’s a super long and complicated term, but what it’s basically a short little sequence found beside the target DNA around three base pairs long that tells the Cas-9 protein to CUT HERE!, instead of accidentally cutting where it isn’t supposed to.

Normally, the DNA would try to repair itself by trying to bond random base-pairs together. Instead, scientists capitalize on this by adding DNA primers that act as a guide for which base-pairs will bond. This way, scientists ensure that their edit will be made. Et voila! We have an edited strand of DNA.

Cas9 protein cuts the target DNA. (Photo from “Expanding the Biologist’s Toolkit with CRISPR-Cas9” by Samuel H. Sternberg and Jennifer A. Doudna)

One of the biggest downsides of CRISPR is unintentional, or off-target genome editing. Because the DNA goes through a “double-strand break” at the PAM, there is a possibility for unintentional and potentially dangerous edits to be made to perfectly normal segments of genetic code since there is no concrete guide for the cell to follow when it repairs itself. Scientists just hope that the primers do what they are supposed to.

For example, take these two sequences:

…CTCGAGGAG…

…CTCGTGGAG…

You may think that they aren’t that different. You may not even have caught the difference between the two.

Well, the first sequence is the normal sequence, noted by the little bit ‘GAG.’ The second sequence has one letter difference, ‘GTG.’ This one letter difference may seem small, but this mistake causes a disease called sickle cell amenia, a disease that causes the formation of unhealthy blood cells that can’t carry enough oxygen around your body.

If this sequence is accidentally translated and permanently edited into the DNA, the consequences are huge. Mistakes like these are why CRISPR, though incredibly accurate, is still prone to mistakes

Luckily, prime editing solves this problem.

PRIME TIME: What is prime editing?

Prime editing is a new technique discovered by researchers at the Liu Group. It is a true, “search-and-replace” technique that allows you to edit and replace specific segments of DNA. Think of it as a find and replace shortcut on a Microsoft Word document: you click Ctrl + H to search for any word you want, then type in what you want to replace that word with. At a macro-level, this is how prime editing works.

Look at all those random dots and lines. Actually, not random: it’s really, really cool. (Photo from Science).

(I literally freaked out when I read the research paper because this is SO COOL.)

More importantly, Liu and Anzalone found a method that allows you to edit DNA without a double-strand break or a donor DNA templates, eliminating the possibility for off-target mutations and unintended editing, one of CRISPR’s biggest downfalls.

The nitty-gritty stuff — how prime editing works

Prime editing has some similar elements to gene editing using the CRISPR-Cas9 method: prime editing also uses the Cas9 protein, along with a guide RNA (known as a prime editing guide RNA, or pegRNA). The mechanism of the guide RNA locating the Cas-9 protein to the target DNA is the same, too.

However, there are two key differences between CRISPR-Cas9 and prime editing: a modified Cas-9 protein and a reverse transcription enzyme.

Those were a lot of big words, let me break it down.

Like the CRISPR-Cas9 method, the DNA is unzipped and the pegRNA will bond to both sides of DNA. In the original CRISPR-Cas9 method, the Cas-9 protein acted like scissors and cut both strands of a piece of double-stranded DNA. In prime editing, researchers have modified the Cas-9 protein to only nick one strand. As a result, a tiny little flap is created on one strand of DNA, while the other is completely intact.

pegRNA (green) has bonded with one strand of the target DNA (blue). The other strand has been nicked, creating a tiny flap that is a few base-pairs long. (Photo from “Search-and-replace genome editing without double-strand breaks or donor DNA” by Andrew V. Anzalone et al.)

Now, it’s time for the reverse transcription enzyme to shine. For the desired edit to be transferred from the pegRNA to the target DNA, the reverse transcription enzyme reads the RNA and finds the complementary base pair, creating the new, edited segment of the DNA.

The pegRNA bonds with both strands of the target RNA. The reverse transcription enzyme with the desired edit is located on the same strand as the flap. Complementary base-pairs are bonded to the pegRNA segment with the desired edit. (Photo from “Search-and-replace genome editing without double-strand breaks or donor DNA” by Andrew V. Anzalone et al.)

Awesome! We have one strand of edited DNA, but we aren’t out of the woods yet! We still have two problems, one of which is that pesky little flap. Luckily, another endonuclease in that cell will snip it away.

The flap is cut off by a different endonuclease, leaving just the DNA. (Photo from “Search-and-replace genome editing without double-strand breaks or donor DNA” by Andrew V. Anzalone et al.)

Problem one: solved.

Now, we have two strands of DNA but only one side has the edit on it. Remember, DNA only bonds with its complementary base pair (A bonds with T, and C bonds with G), so if the two sides are mismatched, the strand of DNA can’t bond or be rezipped.

To solve this problem, a different guide RNA guides another Cas-9 protein to the unedited strand and cuts the mismatched section off. This encourages the cell to use the edited sequence as a guide for repairing the section of DNA. Once the cell bonds the complementary base pairs to the edited strand of DNA, the edit is now complete.

The mismatched segment on the bottom strand has been cut away, allowing the cell to repair the DNA using the top strand as a guide. This completes the edit. (Photo from “Search-and-replace genome editing without double-strand breaks or donor DNA” by Andrew V. Anzalone et al.)

Problem two: solved.

Now, all we’re left with is a precisely edited segment of DNA with practically no chance of unintended edits and mutations. Plus, prime editing has the power to edit way more precisely than CRISPR-Cas9 could.

TL: DR

To recap, prime editing using these seven steps:

  1. The pegRNA locates the target DNA and the target DNA is unzipped.
  2. The Cas9 protein cuts a tiny flap in one strand of the target DNA
  3. The pegRNA bonds to the target DNA. Keep in mind, the flap is still just hanging there.
  4. The reverse transcription enzyme adds the desired edit to the strand with the flap.
  5. A natural endonuclease in the cell cuts of the little flap.
  6. The DNA tries to rezip itself, but the edit and the original DNA won’t bond because they aren’t complementary bases. So, a separate pegRNA and Cas9 team locate the strand of original DNA and cut it off.
  7. The cell repairs itself, using the edited strand of DNA as a guide.

There you have it, an edited segment of DNA!

Will prime editing take over CRISPR?

Though prime editing is extremely cool, we aren’t sure for certain that it is the be-all, end-all solution to editing genetic diseases or gene editing in general. This method was only introduced this year, so we can’t know the long-term effects of prime editing; even though prime editing is more precise, CRISPR has been known and used for longer.

Still, it is fun to think of all the applications of prime editing (because they are endless).

Let’s sum this thing up

For those of you who love to skim, here are some key takeaways of prime editing:

1. Prime editing is an upgraded version of the most common gene editing technique, CRISPR.

2. It makes edits without undergoing double-strand breaks, meaning there is practically no chance that off-target edits occur.

3. Think of prime editing as the find-and-replace shortcut on Microsoft Word, but for your DNA.

According to the Liu Group, prime editing could correct around 89% of all known human mutations.

Imagine. That. Diseases like sickle cell diseases or Tay-Sachs disease could be cured. And as more research and tests are done using prime editing, who knows what we have the power to edit.

Innovations and discoveries like these continue to bring gene editing to the spotlight, and honestly? I couldn’t be more excited.


Hi! My name’s Eliza and I’m a 15-year-old Innovator at The Knowledge Society (TKS). I love gene editing and I’d love to hear your thoughts / ideas on the topic! Connect with me on Linkedin or subscribe to my newsletter.

Thanks to Freeman Jiang

Eliza Aguhar

Written by

Gene Editing Enthusiast & Researcher | Global Sustainability & Climate Change Activist | Innovator at The Knowledge Society (TKS)

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