The Breakthrough Player in the Genetic Research Field: Prime Editing.
Word on the street is that, in theory, Prime Editing could essentially correct up to 90% of all disease causing genetic variations. That’s quite the statement.
So, before we get ahead of ourselves, what is Prime Editing and why do the experts think that it could change the gene editing field?
Prime Editing is a method of gene editing in which individual DNA letters can be changed, deleted, or added, without drastically damaging the DNA strand itself. This is done through precisely slicing just one strand of the double helix. It’s quite the upgrade to the previous CRISPR-Cas9 systems, which originally played key roles in motivating exploration into gene editing.
First, let’s talk about CRISPR.
The CRISPR-Cas9 system created ripples in the gene editing field, making the pathway to editing animal and human genes a little bit clearer. It allowed researchers and laboratories all over the world to gain more insight into genetic diseases and mutations, and realistically explore ways which gene editing technology can be applied to our modern day society.
Yet despite all of its fame and glory, CRISPR-Cas9 had some undeniable faults.
“Genome Vandalism”
It has been known to be a little bit ‘off target’ when it comes to unintentionally cutting sections of the DNA (breaking both strands of the DNA’s helix), often times interrupting cell function and damaging vital systems, (which is not exactly ideal).
A little background info on CRISPR
(if you know all of this, feel free to skip ahead to the Prime Editing section down below).
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR, commonly pronounced as ‘crisper’, is a short form of the full name CRISPR-Cas9.
CRISPRs are specialized stretches of DNA, and Cas9 is an enzyme that takes on the role of a really tiny pair of scissors that snips DNA strands.
CRISPR technology was originally inspired by the defense processes of bacteria and archaea. Such organisms skillfully used CRISPR-derived RNA as well as Cas proteins in order to halt viral attacks as well as foreign bodies, and their entry into DNA. This is made possible through locating, cutting, and destroying the DNA of any intruder. Adapting such techniques, researchers have been able to apply the same technique towards gene editing of much larger organisms, on a much larger scale.
The Cas9 enzyme and the guide RNA (gRNA) of CRISPR-Cas9.
The system consists of two key molecules that introduce the mutation into the DNA.
The Cas9 enzyme is, (as I like to think of it), a super tiny pair of scissors, which will cut the two strands of DNA at the specified location in the genome, making room for DNA to be added or removed.
This molecule works hand in hand with the guide RNA.
The guide RNA, more commonly known as gRNA, is a long strand of a predesigned RNA sequence, along with an RNA scaffold. Essentially, the scaffold part will bind to the DNA, and the predesigned sequence will guide the Cas9 to the correct section of the genome which needs to be edited.
Key Concepts:
The guide RNA has a sequence of bases which are designed to be complementary, or the ‘correct fit’ for the targeted sequence which will later be edited in the genome. (In theory, this means that the gRNA would bind only to the target sequence, yet in application, this is not always the case.)
Generally, the guide RNA consists of a sequence of 20 bases. These are all fitted to the targeted sequence of the gene that will be edited. However, the issue arises because not all 20 of the bases need to be matched for the guide RNA to decide to bind in a particular location. For example, if 19 of the bases in the guide RNA happen to be located in another place in genome, there is the possibility that the guide RNA will bind there instead of the intended sequence, resulting in the Cas9 enzyme cutting and introducing a mutation in an entirely incorrect location.
Here are some of the basics of genome editing, in case you needed a refresher :)
The genomes of an organism are essentially packages of messages and instructions within their DNA sequences.
Therefore, genome editing is the process of changing or altering these sequences, and changing their messages. This can be done by cutting, or making a break in the cell’s DNA, sort of tricking it into making the changes one wants to make.
Once the DNA is cut, the cell’s natural repair mechanisms will kick in, and add the intended mutations to the genome.
There are two main ways in which this process occurs. The first would be non-homologous end joining, which in simpler terms, means gluing the two ends back together. However, this method has been known to introduce many unwanted errors, as nucleotides can be accidentally introduced or deleted in the process, creating disruptive mutations.
The second method involves fixing the break by filling in the gap using a sequence of nucleotides. The cell uses a DNA strand as a template, which scientists can often supply, making the specific changes which they wish.
(Or, less productively, the gap can be filled by randomly grabbing nucleotides from around the cell.)
Prime Editing builds onto the CRISPR editing techniques, notably resulting in much more accurate and clear results.
Using the same Cas9 nuclease that’s deployed in the CRISPR system, Prime Editing enhances the molecule with ‘superpowers’. Through combining the enzyme with a guide RNA known as pegRNA and a reverse transciptase, Cas9 can get to it’s desired location in the genome and add the sequence with minimal error.
“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,” said Liu. “All will have roles…This is the beginning rather than the end.”
So, let’s get to Prime Editing!
An engineered Prime Editing Guide, known as a pegRNA, encloses the target site, as well as the desired edits to the genome. This engages the prime editor protein, which is built up of the Cas9 nickase that’s fused to a reverse transcriptase. The Cas9 nickase component of the protein is guided to the targeted DNA site by the pegRNA. The reverse transcriptase domain then uses the pegRNA to template the reverse transcription of the edit, and polymerizes DNA directly onto the indicated DNA strand.
The edited DNA then replaces the original strand of DNA. This creates what is known as a heteroduplex, which contains one edited strand and one unedited strand. The editor will guide the resolution of the heteroduplex to look to copying the edit onto the unedited strand, and the process is finished.
The Prime Editor’s intentional modifications to the CRISPR system:
- The fusion between the Cas9 and the reverse transcriptase was intentional, minimizing the number of individual components which would be entering the cell, (less room for error).
The Prime Editing guide RNA (pegRNA):
- It is a guide RNA which encodes the RT template, including the desired edits. The PBS, or the primary binding site, is essentially the sequence which was complementary to the nicked genomic DNA strand, serving as a vital point of initiation in order for reverse transcription to occur.
Heres what I think is the coolest part:
In order to resolve the mismatched DNA so that it can favour the edit, the Liu lab turned to a former base editing strategy. Because the original sequence on one strand and the edited sequence on the other strand do not match, they began nicking the unedited strand, making the cell repair by using the edited strand as a template or reference for what the nicked strand should look like.
So that sounds interesting, but why is it important?
If you’re not too interested in the science behind Prime Editing, it can sound kind of like mumbo jumbo (at first, it did to me!). Yet, after learning about the possibilities and full capabilities of the technique, I couldn’t stop looking through the research.
Aside from the exciting developments made from the original CRISPR model including cutting one single DNA strand (which stops the former error of prompting the cell’s problematic repair system), Prime Editing is one of the most versatile and flexible gene editing tools in the tool box.
Before Prime Editing gloriously emerged, there was a consensus among researchers that because of CRISPR’s limited and somewhat risky methods, there was a distinct CRSIPR tool that would be required for each form of editing: one for deleting, one for adding, and one for substituting. And yet, Prime Editing can preform all of these tasks itself, seemingly without breaking a sweat. What does this mean? It means less equipment, lower costs, and fewer obstacles to slow down research!
Given that Prime Editing can swap DNA letters…
It could in fact treat quite a large array of inherited diseases in the near future, (nearly 7,000 to be exact).
Sickle Cell Disease: A fix requires switching a T to an A at a specific position.
Tay-Sachs Disease: A fix requires removing the 4 unnecessary letters of code.
** Recall: the four letters of the DNA alphabet are A, C, T and G
Liu’s team has tested Prime Editing on neurons from mice and human cells so far, with the rate at which additional locations were unintentionally edited being quite low, at roughly 10%.
But let’s not get too excited,
There’s still lots of work to be done before Prime Editing reaches it’s full potential!
Although things are looking good, its still quite a new technology, and warrants many more additional studies before widespread usage and distribution. The Liu lab emphasized the importance of taking further steps to observe and test the capabilities of Prime Editing, including investigation of inadvertent effects on the cell, and usage in different cell variations.
“A major aspiration in the molecular life sciences is the ability to precisely make any change to the genome in any location. We think prime editing brings us closer to that goal. We’re not aware of another editing technology in mammalian cells that offers this level of versatility and precision with so few by-products,” stated Liu.
(As well, it’s important to address the ethical concerns which arise as a result of gene editing, and the public opinion of such practices are often polarized when it comes to altering the genes of infants for immunity.)
Key Points:
- Prime Editing was built off of many procedures and techniques which are well established in CRISPR editing methods.
- Although CRISPR was revolutionary in itself, it did have it’s own faults, which Prime Editing intends to solve.
- Prime Editing could very well play an integral role in minimizing genetic mutations and diseases in the future, as shown through Sickle Cell Anemia and Tay-Sachs disease testing.
- Prime Editing is incredibly versatile and exciting, yet it is still quite new and requires further research to understand the full scope of it’s capabilities.
