A world where all diseases have cures and where we are able to design the genetically perfect child. It is no longer a dream, but rather a reality thanks to the revolutionary technology of Prime Editing.
Prime Editing is a genome editing technology that writes new genetic information and replaces it in specified DNA sites. The way Prime Editing works is by using Cas9 attached to a prime-editing-guided RNA (pegRNA) along with a reverse transcriptase.
Before detailing the process of Prime Editing, it is important to understand each component and its function.
Cas9 stands for CRISPR associated protein 9, and it is an enzyme that is mainly used to cut DNA in Prime Editing. PegRNA (Prime Editing guided RNA) is RNA that is used to locate the targeted section of the genome, based on the order of the nucleotides. Reverse transcriptase is an enzyme used to create DNA from an RNA template to convert the old, unedited to new, edited DNA.
The process of this genome editing is, the Cas9 makes a nick in one side of one of the strands of DNA. The reverse transcriptase then binds the new, unedited DNA to the pegRNA, allowing it to be converted and edited. Now one of two things occur. Firstly, the new, edited DNA is bound to or overlapped with the old DNA — which is now not attached to the double helix on one side — converting it. Then, it is reattached to the strand and the nick is automatically repaired. Secondly, the old, unedited DNA — in one strand — is completely cut off and the new, edited DNA is inserted into its spot.
After the previous process, one strand of the double helix is fully edited. To edit the other strand, the Cas9, guided by pegRNA, makes a small cut in the unedited strand of the double helix. The cut causes the cell to remake that section of the strand, using the edited DNA on the opposite side as a guide. As a result, the targeted sections of both strands are fully edited.
Imagine you are restoring an old broken down plane. Firstly, you would remove the damaged parts of one side of the plane. In Prime Editing, this is performed by the Cas9. Next, you would trim the material for the new wall to fill in the hole in the plane. This part is performed by the reverse transcriptase, which binds the new, unedited DNA (the new material) to the pegRNA (the size of the plane).
Following that, you would insert the new material into the hole in the plane. For the next couple of steps, imagine that there is a small hole in the opposite side of the plane (cut in the unedited strand of the double helix). This would be done by the Cas9 guided by pegRNA.
To fix the hole, you would use the new material and insert it into the small hole. That is also done by the Cas9. However, when you are fixing the hole in the plane, you are using the opposite side as a guide. This leads you to notice that one side has new material while one side still has the old material, so you fully replace the other side with new material as well. This is done through the cell naturally. While it is repairing the cut in the unedited strand, it is referring to the opposite strand, which is completely edited. This leads the cell to edit the original strand as well, overall completely editing that section of the DNA.
The Importance of Prime Editing
Prime Editing allows for more precise and safer DNA edits. One reason for this is, Prime Editing only replaces/converts one strand of the double helix, and relies on the property of the cell to duplicate it to the other strand. This is important because it minimizes the chance of dangerous mistakes. David Liu, a biologist and chemist who focuses on DNA editing, says that Prime Editing searches for the targeted DNA and precisely replaces it.
Prime Editing can treat countless more diseases than previous DNA editing technologies like CRISPR. One example of these diseases is inherited diseases, such as sickle cell disease and Tay-Sachs disease. This is because using Prime Editing, doctors can swap DNA chemicals with each other. Prime Editing can be applied to edit and cure cells that do not divide because it can correct the changes in large populations.
Prime Editing is mentioned as an extremely precise process since it can remove up to 80 chemicals in the genome in a given spot.
Using all of the above points, Prime Editing can correct 89% of all genetic variants that cause diseases.
Prime Editing achieves lower rates of unwanted nicks and changes in the DNA, as well as more versatile opportunities for edits.
“The versatility of Prime Editing quickly became apparent as we developed this technology. The fact that we could directly copy new genetic information into a target was a revelation.” Dr Andrew Anzalone at the Broad Institute of MIT and Harvard
An example of these versatile changes as mentioned in a published research paper by David Liu and his team is, using Prime Editing, they could make single-nucleotide changes, changes where there were too few nucleotides, too many nucleotides or any combination of these.
A nucleotide is the single building block of DNA and RNA. It is made of one of the four chemicals or letters, with one molecule of sugar and one molecule of phosphoric acid. Changes in the concentration of these nucleotides cause different diseases. As mentioned before, Tay-Sachs disease is curable through Prime Editing and Liu’s team accomplished that by removing the four DNA chemicals that caused it.
Prime Editing vs. CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is the cell defence system that builds the foundation of this gene therapy.
The way CRISPR works is it uses Cas9 directed by RNA, to cut out the targeted parts of the DNA on both strands. The damaged or unedited parts of the DNA are then either edited, which is called homology-directed repair, or it is removed. If it is removed, the Cas9 and RNA either patch together both ends of the cut, which is called nonhomologous end joining, or it inserts completely new genetic material.
Researchers focus on homology-directed repair, as it is more reliable and more precise. CRISPR can also be performed by cutting out the damaged section of the DNA, and leaving it open for the cell to naturally regenerate the DNA. However, this method is unreliable and can lead to unintended inserts or deletes of DNA letters.
The way the guide RNA works in both CRISPR and Prime Editing is by scientists first identifying which part of the DNA is causing the disease, or what part they need to edit. They then identify the exact chemicals or DNA letters in the genome that they need to fix. The scientists then create a guide RNA that matches the exact order of the part of the genome. The guide RNA then is attached to Cas9 and searches through the entire genome until it finds the targeted area.
Prime Editing is a more advanced CRISPR tool. It uses the same technology as CRISPR, plus, prime-editing-guided RNA (pegRNA), and the reverse transcriptase.
Prime Editing only uses the Cas9 to cut one part of one strand of the DNA, whereas CRISPR cuts both strands. Due to this aspect, Prime Editing is more precise and more reliable. Cutting both strands can also lead to more collateral damage. While using Prime Editing, apart from the first cut into one strand, there is very little room for error because instead of replacing or deleting the DNA, it converts the old nucleotides into the new, edited ones.
One of CRISPR’s methods relies on the cell’s DNA repair system, not allowing the researchers to make the edits they want.
Due to the differences in the processes of CRISPR and Prime Editing, CRISPR is unable to directly convert one DNA letter into another one. However, Prime Editing can not only achieve that but also cure diseases caused by multi-letter mutations, like Tay-Sachs Disease.
As you know, when using CRISPR, donor DNA is required to be inserted in the gap in the double helix. While using Prime Editing, no donor DNA is required throughout the entire process. This is because the reverse transcriptase creates new, edited DNA based on the pegRNA and the opposite strand is naturally converted by the cell.
Think of CRISPR like a fairly basic swiss army knife. The components are very basic and in total there are only three or four different changes. Now imagine Prime Editing as an extremely high-quality, expensive swiss army knife. This is because, as mentioned before, Prime Editing is extremely versatile. Along with that, it can make several different types of changes, directly convert old DNA and make specific DNA letter swaps.
To summarize, Prime Editing can achieve everything that CRISPR and base editing can, including multi-letter genome editing with more precision, less collateral damage and lower risk of unwanted changes.
Prime Editing in the Future
DNA editing is one field that is sure to expand over the next couple of decades.
With technologies like Prime Editing, editing DNA to prevent diseases is extremely feasible. As mentioned before, diseases that are inherited can be treated currently by removing or replacing the nucleotides that cause those diseases.
In the future, several other diseases can be treated using Prime Editing. Take the example of sporadic cancer, which means the cancer is not hereditary. All types of cancer are caused by mutations or damages to genes, which causes abnormal developments in the body or it stops the body from working correctly. One way that Cancer could be treated, by using the same principles as today, is to replace the mutated genes. One of the genes that prevent tumour growth is p53. In the case where someone has Cancer, one of the main causes is defective or damaged p53 genes. If doctors could replace those genes, it would tremendously help with the treatment. Speaking of Cancer genes, the other three main types of genes that cause Cancer are Oncogenes, Tumour Suppressor Genes and DNA Repair. In the future, if we use Prime Editing to either restore or replace these genes, we will be able to cure certain types of Cancer.
On the subject of curing diseases, another application of Prime Editing is to prevent diseases from being passed down from parents to children. This is currently being tested in the instance of preventing HIV to children by using gene therapy on the embryos. In the future, this field will only grow bigger.
In the near future, scientists will be able to use Prime Editing to help patients recover and be used as a treatment for several diseases.
When discussing long term applications, it is important to think about what Prime Editing could do, not for humans or animals, but also machines. Artificial Intelligence, which includes Machine Learning, Deep Learning and more, uses artificial neural networks. These neural networks are based on the human brain. When Prime Editing can be publicly used on humans, it would open up thousands of possibilities for machines to replicate Prime Editing and create a virtual version. This could be used for self-repair, to fix a bug in the main operating system’s program. Once that is established, it can be used in every single piece of technology in the world, resulting in devices that do not need to be replaced, self-updating machines and more.
One more commonly discussed application of gene therapy, specifically Prime Editing, is editing genes to create the “perfect” child. This concept revolves around the idea of applying the processes mentioned above into the embryos of humans. Prime Editing of embryos could be used to correct disease genes or to offer protection against infections like Alzheimer’s. Protecting from diseases and even aspects of humans that decrease as we age, is an extremely underdeveloped topic. Dr. Luhan Yang, a Harvard recruit who helped develop CRISPR, says that this very concept could be used to reverse ageing in the future.
Several projects are underway around the world that are experimenting and refining the idea of genetically editing human embryos to avoid disease, control the attributes of a child and more. Using CRISPR, doctors have said that editing human embryos is not particularly hard. However, there are limitations and extreme risks when using CRISPR. Guoping Feng, a biologist working on a similar project said that the efficiency of using CRISPR to edit or remove a gene is only 40% while using it to make specific DNA letter swaps, is only 20%. Prime Editing can achieve everything CRISPR can, but better and more reliably, including DNA letter swaps. Therefore, theoretically, we should have everything we need to be able to complete these trials and release this technology to the public.
Not quite. While this technology is extremely interesting and promising to some people, others are thoroughly disturbed and think it is a violation of ethics. (The next couple of lines are referring to all the future applications of Prime Editing, not just embryos editing). Dozens of countries have prohibited the practices of this technology because it violates human rights. Several scientific societies and governments have agreed that there is too high of a risk to put not only current human lives at stake but also each of the test subjects’ children and grandchildren. The European Union Convention on Human Rights and Biomedicine decided that performing gene therapy on sperm cells, eggs and embryos violates human rights and is prohibited.
While there are thousands of potential applications of Prime Editing, only time will tell the fate of this technology.
Prime Editing is an extremely powerful genome-editing tool that adds to the traditional method of CRISPR. It is able to perform DNA edits more efficiently while decreasing the risk of unintentional changes and mutations. As Prime Editing continues to evolve, it has the potential to redefine life as we know it.