Biology’s Successor: Prime Editing
Some peers are texting each other on a summer day, bored out of their minds, obeying the Stay at Home order of California.
What could we observe of their dialogue?
Let’s check it out.
Introducing C.R.I.S.P.R.
We notice that a peer is pondering the topic of prime editing, the latest of what CRISPR has to offer. You may be familiar with CRISPR, a genome-editing tool introduced back in 2013:
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R epeats
CRISPR is the widely renown genetic “snipping” tool. Some biologists even refer to CRISPR as a “Swiss Army knife” rather a pair of scissors.
What is prime editing?
Prime editing is different from CRISPR, but also is derived from it. The first-generation CRISPR 1.0’s scissor component called Cas9, is a protein that will cleave foreign DNA within a cell. This protein is the one of the most important components of the whole process of genome-editing. It “snips” out specific target sequences in DNA, which will drive out mutations in that cell completely. Prime editing takes this process further: after tampering with DNA, it will be replaced with the corrected sequence and will allow the cell to replicate this newly injected sequence. For example, prime editing may simply change a “C” in a DNA sequence to a “T.”
Understanding the process of CRISPR
Engineers program and implement on things called nucleases. A nuclease is an enzyme that severs nucleic acids. These types of enzymes belong to a class called hydrolases, and for this particular study, ribonucleases act upon RNA, while deoxyribonucleases act upon DNA. These enzymes are found in both plants and animals alike.
Nucleases are also described as restriction enzymes, in which there exists three important types:
Type I: splits a targeted DNA molecule at a random site
Type II: splits a targeted DNA molecule at a recognized site
Type III: splits a targeted DNA molecule at a certain distance from recognition site
Type II and Type III restriction enzymes are used indefinitely in genetic engineering. Another name for a restriction enzyme is a restriction endonuclease, which is a protein produced by bacteria. This protein is extracted from bacteria to then orchestrate the elimination of unwanted letter typos within DNA, especially certain genes.
Bacteria defend themselves by using these special enzymes to go up against bacterial viruses, better known as bacteriophages. A phage’s DNA will be replicated once it enters one of its host cells, but these enzymes will restrict or limit infection by snipping its DNA into many pieces. This process is conducted by the enzyme’s catalyst properties; it will add a water molecule to a chemical bond to split it (in chemical terms, this is hydrolysis using a catalyst).
The endonuclease has a sidekick called the methylase; these enzymes protect recognition sites of their DNA from the endonuclease by adding methyl groups to adenine or cytosine bases which are located in the site.
Nucleases are created by using Cas9, a protein that will use crRNA (containing the foreign DNA and its complementary pair of that subset), to interact with the PAM sequence in foreign DNA. This Cas9 complex will bind tighter to the sequence carrying the complementary sequence to the foreign DNA, so it can be cleaved shortly after.
A deeper dive into prime editing
Prime editing is a method that is bound to be endorsed by the world.
How exactly does prime editing work?
A new and improved Cas9 targets DNA using a guide RNA. This guide (engineered) RNA finds the site, containing the DNA sequence (which must be severed and replaced) and hybridizes to the site.
The nick made by the RNA will expose a three-inch hydroxyl group (shown in Figure 1c) which is used to prime or prepare the reverse transcription (from RNA to DNA).
The RNA will replace the severed-off sequence, thus inducing the cell to replicate healthy DNA and restore healing of the infected cell.
With correspondence to Figure 1c, this process results in two flaps of DNA: one with a measure of three inches, another with a measure of five inches. The three-inch flap will contain the edited DNA while the five-inch flap contains the unedited DNA. The hybridization of the five-inch flap is expected to be thermodynamically favored (in chemical terms, this process will occur on its own, paced at a certain time).
This is how prime editing differs most from CRISPR 1.0: one strand is left unedited, preventing more mutations to occur and creating more accurate incisions. Another differentiation between CRISPR and prime editing is that CRISPR involves snipping while prime editing is a tactic of searching and replacing of the human genome.
Keep this in mind: FEN1 is a type of endonuclease that helps with metabolism and EXO1 is type of endonuclease existing within humans that characterizes activity in a cell.
The five-inch flap is the preferred surface of FEN1 to be able to excise and repair sequences, where EXO1 will remove superfluous DNA.
The last steps of repairing mismatched polynucleotide strands would be a process that the cell will do on its own: the cell will permanently reciprocate the edited DNA to replace the unedited DNA by creating a complementary strand of the edited DNA.
To ensure that the edited strand will be copied efficiently, an additional nick to the unedited strand will be made to alert the cell that there is DNA damage. Once the cell is aware of the damage, it will copy the newly repaired DNA.
Prime editing and the future
Prime editing is the genetic face of the future. It will change people’s lives in a positive way.
With the ability to point at certain locations on the human genome where certain mutations will occur, it is safe to say that we could also be able to predict where and when diseases could develop. By looking at someone’s genome using display Cas9 screens, we are able to predict 10 or 20 years in advance.
Once prime editing is fully functional, we could also understand RNA expressions, the proteins made as a product from RNA and DNA, somatic cells, and germ cells on a different level of comprehension.
We can also graduate from testing with cells in laboratories to a clinical level, where we could use an actual patient’s cells to cure them of their disease. Cancer, cystic fibrosis, sickle cell disease, Tay-Sachs disease, etc. will be knocked out of somatic cells for good.
Despite ethical issues, prime editing is biology’s next step to a healthier world of people.
“To not kill two strands with one snip.”
— Prime Editing
