Base Editing and Prime-Editing: The Next Step in Gene Therapy
The year was 2012, Jennifer Doudna and her colleagues had just discovered that the guide RNA sequence could be changed to direct Cas9 to any target we want to hit. It’s been 10 years since the massive breakthrough of CRISPR-Cas9, a system for genome editing, the field has been subject to continuous improvements and growth.
Innovation pushing CRISPR one step further is Base Editing and Prime Editing. These two have increased the potential of gene therapy to a whole new level. However, what are Base Editing and Prime Editing? Why are they so groundbreaking and why haven’t we done anything with it? Those are some of the questions I will be answering in this article so get your popcorn out for a deep dive into the world of Gene Editing.
Overview of Cas9 ✂️
Before you can learn about Base Editing and Prime Editing you need to know how Cas9 works and what are its limitations. Of course, Cas9 is a whole article in itself so this is just going to be a good overview for those who don’t know how Cas9 works. Cas9 or you might know as CRISPR can edit the genome of any living thing! Therefore, to put it more simply it’s a Ctrl+F and Delete protein. However, Cas9 isn’t just one type of protein as there are many variations of this protein that vary in size, PAM and bacteria derivation. For example, we are going to look at the most common one which is found in Streptococcus pyogenes.
Cas9 is at the end of the day a protein meaning it doesn’t have eyes. So for Cas9 to be able to find the target sequence we need to give it a guide or in this case a guide RNA (gRNA). Most gRNAs have three main features the first two are crRNA and tracrRNA which the lab turns into a Single Guide RNA (sgRNA). The third feature is in the gRNA sequence itself, it is a specific extension at the end of the gRNA which we call the PAM. It has a 2–6 base-pairs length that needs to match with the DNA or else the Cas9 Protein won’t make a Double-Strand Break (DSB) required for its function.
Deep Dive: Base Editors 🧫
After the overview of how Cas9 works, we can now talk about Base Editors and how they work. Our DNA is made up of four building blocks Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). These base pairs have their match for Adenine its Thymine and for Guanine its Cytosine. However, sometimes the body could create a Single Nucleotide Polymorphism or Point Mutation. This mutation occurs when the body mistakes one base pair for another therefore, instead of putting Adenine with Thymine they put Guanine with Thymine. This type of mismatch could lead to many dangerous consequences such as Cancer, Sickle-cell anemia, and etc.
There are two types of Base editors — Cytosine Base Editor or Adenine Base Editor.
Cytosine Base-Editors
The Cytosine Base-Editors simply turns a Cytosine (C)into a Thymine (T), well not exactly thymine but something close to it called Uracil. There isn’t much of a difference between them as they are both the same in the eyes of the cell. The main two components of the Cytosine Base-Editors are Cas9 and modification to cytidine deaminase. The first component is a variant of Cas9 whose job is to make a single-strand break. The second component is an add-on to the protein cytidine deaminase which simply put turns Cytosine into Uracil and the coolest part is that this is a naturally occurring protein.
You might be wondering if Cytosine and Guanine always pair together how come the Base Editor only changed the Cytosine into Uracil leaving the Guanine untouched. Well, because Cas9 can only make a single cut, it makes a break in the DNA strand. So when the cell reads that section it will see the nick and replace the Guanine for Adenine. Fixing the point mutation just like that.
Adenine Base-Editors
The second is Adenine Base-Editors which are very similar to the Cytosine Base-Editors, but have two main differences. One is that instead of changing Cytosine into Uracil it changes Adenine to Inosine. Now you might be thinking Inosine isn’t part of the four Nitrogenous Pairs mentioned earlier.
That is where the second difference comes into play, as cytidine deaminase is naturally found. In the case of adenosine deaminase, David Liu and the group had to engineer the protein and direct the evolution of Escherichia coli tRNA adenosine deaminase, TadA (ecTadA), to turn it into the adenosine deaminase which is pretty impressive. Besides those differences, it works the same way in changing Adenine.
Prime Editing 🧬
Prime Editing has taken the world of gene therapies by storm becoming the golden standard for genome editing. However, what makes Prime Editing so powerful? Well, it will have to go to the fact it doesn’t rely on Homologous Directed Repair and Non-Homologous End Joining which is the way our body repairs the cuts made by Cas9.
Prime Editing has two main new components, one being the guide RNA, which instead of just carrying the target sequence it carries the new genetic information that will replace the targeted sequence and is called pegRNA. So how does this new genetic information actually turn into RNA to replace the target sequence? Well, that is where the second new component comes in called Reverse Transcriptase. This protein would read the pegRNA and turn it into the RNA that would replace the target sequence. The main issue of this would be that once everything was done there would still be a flap formed between the edited DNA and the previous DNA that wasn’t cut. The solution to that is that they just nick the unedited strand making it unfavourable compared to the edited strand to the cell.
Challenges 🚧
There are still many challenges with Base Editing and Prime Editing. One big issue is off-target effects that could cause serious errors as gene binding still isn’t accurate enough. Constitutive overexpression from strong or leaky promoters can result in undesirable outcomes such as cellular toxicity and off-target effects. Another obstacle is the size of the proteins as many in vivo methods of delivery have a limit to how much they can hold. Due to Prime Editing and Base Editing being too big there are some workarounds that are being developed but they are decreasing efficiency making the improvement basically impossible. Lastly, the target sequence itself can sometimes be unreachable, either the PAM is restricting them or the range of the target sequence is too long, making it improbable to cut all of it by Cas9.
There is Still Hope ✨
Even though there are obstacles between now and 100% efficiency, at the end of the day they are problems that are meant to be solved. Labs and countless researchers are finding new ways to push Cas9 above and beyond expectations, with recent discoveries on how to make it so Cas9 doesn’t need a PAM anymore or modifications to the gRNA to improve its target efficiency. Who knows, maybe in the next 10 years Gene Therapies will become just as common as prescribing Advil.
