Base Editing, or How to Edit the DNA Without Cutting It

As you know, our DNA is composed of 4 nucleotides called Adenine (A), Guanine (G), Thymine (T), and Cytosine (C). Now these are not the only existing nucleotides. For example, in our RNA, there is no thymine, and instead, we can find another nucleotide called Uridine (U). What’s interesting about these nucleotides is that their structures are quite similar. For example, Uridine looks pretty much like Cytosine. The only difference between them is that Cytosine has an amino group whereas Uridine has an O2 group.

Now, remember we said we were going to use a deaminase? What do you think this enzyme would be able to do? Yes, you guessed right, you brilliant geek! A deaminase removes amino groups.

Here’s the bonus points question: What would happen if this deaminase found a Cytosine nucleotide and removed its amino group? If you said that Cytosine will be converted to Uridine, you deserve a cookie!

Okay, so by binding a deaminase to dCas9, we can bring this complex, which we will call a base editor, to a specific DNA site. The deaminase can recognize a Cytosine nucleotide within the target sequence and convert it into Uridine. But what effect will that have?

If you remember, we said before that Uridine replaces Thymine in the RNA. And our cells know that very well. In fact, they know it so well, that when they find a Uridine nucleotide in the DNA sequence, they don’t really freak out. They’re more like “oh, somebody put a Uridine here instead of a Thymine, typical… It’s fine, I’ll fix it.”

Do you realize what just happened? We tricked our cell! By deaminating Cytosine into Uridine, we made the cell think that there was a Thymine there, and now there is one! So we’ve effectively replaced a Cytosine with a Thymine, modifying the genetic sequence! That’s what we will call a C-to-T edit.

Go ahead and tell us that’s not awesome, we dare you!

Different base editing tools can introduce various edits

Okay, now that you have an idea of how base editing works, let’s discuss what we can actually do with it. As we said, the first base editing experiments substituted a Cytosine with a Thymine. That’s because the deaminase they bound to dCas9 was a Cytosine deaminase, which means that it can only deaminate cytosine. But what would happen if we replaced that Cytosine deaminase with another enzyme? As you might have guessed, we would be able to introduce other types of edits.

The second type of base editors that came out used an Adenine deaminase. Interestingly, when we deaminase Adenine, it turns into another nucleotide called Inosine. And while Inosine is not in our DNA or RNA, it is also something that our cell recognizes frequently. Interestingly, Inosine can be found on our DNA when a Guanine nucleotide has been damaged. So, again, we’re tricking our cell. We’re making it think it has to repair a damaged Guanine nucleotide, when it was an Adenine all along!

So that’s two types of edits we can make, C-to-T and A-to-G. It doesn’t seem like a lot, but what’s interesting is that more than 60% of the disease-causing mutations we can find in humans are either G-to-A or T-to-C. That means that with only these two types of base editors we could already potentially cure a great majority of genetic diseases that are caused by point mutations. Not bad, is it?

In addition to that, some other types of base editors have been developed to target rare types of mutations. This way, C-to-G edits, as well as simultaneous C and A edits are now also possible.

Base editing has limitations

Okay, so far we’ve told you how cool this new base editing technology is. But this wouldn’t be us if there wasn’t a twist somewhere along the line. So, what’s the matter now?

Luckily, this is not as dramatic as in previous episodes. Base editing is indeed an awesome technology. However, some limitations have been found in the last years. But worry not, because this is the bread and butter of research scientists!

Bystander Effect: The first and most important limitation of base editors is the bystander effect. That may sound similar to the off-target effects we talked about recently, and you would be right. In this case, however, the effect is not far away in the genome, but very close. In the same target sequence, in fact.

Because what we told you about above was a perfect example, where the deaminase finds and edits a single Cytosine nucleotide in the target sequence. But what happens if there are two Cytosines there? Or more? Well that’s where bystander effects come into play.

Most times, we’re going to want only one of the Cytosine to be edited, but the deaminase is not that smart, and it may also modify the other Cytosines around it, resulting in a sequence we did not want. To avoid this, new base editors have been developed that have smaller “Editing windows”, that is the range of nucleotides that the deaminase can see.

Edit size: The other main limitation of base editing is that it can only be used to introduce single-nucleotide edits. While those are extremely important and have many applications, other gene editing strategies require substituting several nucleotides, or even inserting large DNA sequences at a target site. For those strategies, base editing cannot be useful.

Off-target effects: The last limitation we will discuss are off-target effects. Yes, base editors can also recognize the wrong sequence and edit it. While the damage will, in most cases, not be as bad as with regular CRISPR, the wrong edit at the wrong location could still have dramatic effects, so better to reduce those! And interestingly, base editors have raised another issue: since deaminases are not great at differentiating DNA and RNA, base editors can also have off-target effects in the RNA. We’re not going to go into detail on this point just yet, but this was kind of a big deal and new editors have reduced deaminase activity in the RNA, which has partially solved this issue.

As you are reading this, we are sure we are all vibrating on the same line “ this technology is awesome, can’t wait to see what the future holds!”

Luckily for you, this is not Stranger Things, and you will need to wait only one week to read all about it.

In fact, next week, we will take a step back from the technical side and discuss CRISPR therapies that are close to approval. Don’t miss it, it’s going to be a great one!

Meanwhile, if you’re left wanting more on how base editing works, CRISPR Medicine News published a more extensive and technical Explainer on Base Editing, where you will find your curiosity satisfied!