Three billion years ago, bacteria developed an adaptive immune system. This immune system fights viral infections for the bacteria. In the 1970s, geneticists started focusing their attention on genome editing though it was not before March 2011 that Jennifer Doubha and Emmanuelle Charpentier cracked the puzzle of the RNA and Cas9 complex targeting the viral DNA location. They were the ones who propelled researchers into genome editing — methods through which DNA undergoes specific alterations.
To understand the foundation of prime editing specifically, we must look at its predecessor, CRISPR-Cas9.
What is CRISPR-Cas9?
Well, it stands for Clustered Regularly Interspaced Short Palindromic Repeats. And yes, at first, this made no sense to me, but then I began my research.
Let us briefly discuss what happens in a cell and to its DNA when infected by a disease. When infected, the virus injects viral DNA into the cell. Here is when the CRISPR system comes in. It allows us to extract bits of DNA in the affected areas and even replace them. However, CRISPR itself is a site and mechanism that keeps track of which virus the cell has witnessed. CRISPR also enables the bits of DNA to be passed on to the cell’s progeny (protecting the cell for generations to come). Once insertion is complete in the chromosome, the cell copies RNA, a molecule that replicates the viral DNA. In turn, the RNA binds with Cas 9 protein (which contains endonuclease activity). Bound together, they go through the cell’s DNA in search of sites matching the RNA sequence. Once found, the Cas9, RNA, and DNA form a complex permitting the Cas 9 to make a precise incision in the DNA.
But why is all this important?
Hundreds of thousands of people are affected by a genetic disease caused by a point mutation — a swap in one or very few nucleotide bases that creates a mutation. Experts know that these mutations could be solved if only there is a way to switch a single nucleotide bond (example, swapping Adenine with Thymine in a DNA code).
They have further theorized that through genome editing processes, such as CRISPR, this operation could one day be feasible.
Therefore, CRISPR is a part of groundbreaking biomedical research and could potentially lead to curing an approximated 89% of genetic diseases in the future!
Disadvantages of CRISPR:
CRISPR was deemed inefficient and perhaps not fully reliable due to several factors, the first nuisance being the DNA incision. Ineluctably, this alters not only the virus life cycle but our genome as well. Although this can sometimes come in handy, unfortunately, most point mutations cannot be cured by cutting the DNA. The function of the mutated gene would need to be restored, as opposed to being further disrupted. A second concern associated with CRISPR is that it sometimes created extra cuts in the wrong sections of DNA, therefore being imprecise and unreliable. Lastly, the double-strand breaks it induces are mutagenic (could result in translocations of DNA, p53, tumorigenesis…).
The beginning of base editing:
Chemist David Liu worked alongside some of his students in chemically altering a DNA base without further disrupting the gene. They came up with molecular machines termed “base editors” (used in “base-editing” which a different genome editing technology). Base editors directly reconstruct the atoms of a nucleotide base so that it becomes another base. For instance, atoms of the thymine base would be re-arranged so that thymine turns into a guanine base. However, since thymine and guanine do not form base pairs, the cell must dissolve one of the bases. Liu and his team were able to engineer which base would dissolve by nicking the strand of the unwanted base.
Now that we have a fundamental understanding of CRISPR-Cas9 let us look at prime editing.
How Prime Editing came about:
In short prime editing is a newer genome editing technology that draws upon the CRISPR system but makes use of all the features CRISPR was lacking. When developing prime-editing, researchers necessitated a genome editing technique that could:
1. Edit genomes with either single-strand breaks or no breaks at all
Experimentation originated by trying to make single-stranded breaks more efficient in avoiding donor DNA. Oftentimes, retrieving the required DNA posed a challenge, therefore to make this process more efficient, researchers began fusing nickases to different protein domains. This opened up DNA and encouraged the donor DNA to implement itself.
As for “no breaks”, they work through the aforementioned base editing system. The flaw in the base editing system is that the base pair changes are still limited to: cytosine to thymine, thymine to cytosine, guanine to adenine and adenine to guanine.
2. Have a versatile editing technique
All genetic diseases differ from each other and should, therefore, be treated in different ways. This means that genome editing should be very versatile. The editing should account for:
- Substitution (example: substituting cytosine for thymine)
- Deletion of nucleotides
- Insertions
How does Prime Editing work:
Prime Editing uses the Cas9 nickase protein and the guide RNA that locates the target sites. Since we are dealing Cas9 nickase rather than Cas9, instead of a double-strand break, the Cas9 nickase will simply nick the DNA. The guide RNA will only recognize and target one of the two DNA strands (also known as the “target strand”) and will bind to it. The other “non-target strand” will not be affected.
Another component is the reverse transcriptase enzyme. It reads the target strand’s RNA and makes a complementary set of DNA accordingly. The reverse transcriptase enzyme joins the bind between Cas9 and guide RNA, resulting in the formation of a complex. Once the guide RNA has found the target site, the Cas9 enzyme nicks a single strand of DNA. Now the guide RNA has two RNA constituents: the binding region and the edited region (RNA sequence in charge of coding for the necessary changes). Then, after the Binding region binds to the nicked DNA, the edited RNA sequence is reverse transcribed using the reverse transcriptase. The edited strand is incorporated into the DNA, and the target DNA is fixed with the new reverse transcribed DNA. The original DNA segment has been removed.
This leaves us with an edited strand and an unedited strand. To turn the unedited strand into an edited strand, the guide RNA needs to lead Cas9 to cut it. Next, the newly edited strand acts as a template to repair the nick, consequently completing the edit.
The future of Prime Editing:
Being that prime editing is such a promising newer technology, geneticists are applying it to just about everything. From ameliorating individual letter changes in crops to researching gene mutations in cancer.
Other inventions to be made include increasing the scope of new molecular machines. Doing so could help in lessening undesired off-target editing and increasing efficiency. This is how prime editing can make base pair changes to all twelve possibilities!
One of genome editing’s main goals is to be extended to therapeutic applications but geneticists even thought of human enhancement, whether it be stronger bones or smaller changes such as hair color…
How exciting is it to think of how far genome editing can take us!
Summary:
- The immune system of bacteria (CRISPR), uses the knowledge of all past and pre-existing viruses to induce an incision at the mutated “target” site of the DNA’s double helix
- Geneticists then started looking for a way to minimize unwanted off-target location edits, increased precision in the cut, as well as different ways to substitute viral DNA with a beneficial one. They then created base editing that, although still prone to making off-target changes, was able to make either a single cut or switch base pairs with no cut at all.
- Then came Prime Editing, which, although still very much in the “prototype” phase, proves to take up a prominent place in the biomedical world. It overcame many of the challenges that CRISPR was facing such as the limit of changes in base pairs, efficiency, the need to have donated DNA…
- Prime Editing can be applied to humans as well as plants and crops…
What to take away
- Never underestimate the power of something deemed “insignificant” such as bacteria, since it could lead us to revolutionize technologies!
- Keep up to date with the advances of Prime editing, since, who knows, maybe in a couple of years, there will be no such thing as viral genes!
Timeline:
Here are some articles and resources I have used in my research:
