Prime Editing — The Future of Gene Editing

Mikael Haji
10 min readSep 2, 2020

From the smallest single-celled organisms to the largest creatures on Earth — every living thing is defined by its genes.

As a society, we have reached an era in which gene editing is the most efficient and effective way to engineer DNA by replacing, deleting and modifying a genome of a living organism. Nonetheless, It is extremely important that we identify some of the most accurate technologies in gene editing in order to improve the process and maximize its efficiency.

New “Prime Editing” Method Makes Only Single-Stranded DNA Cuts

Basic Knowledge

Before we get into the actual content of the article, below are a few pieces of information that you should know.

  1. What is a Genome? A genome is the entire genetic code of an organism including an organism’s genes and the Deoxyribonucleic acid (DNA) code it is made of.
  2. What is the Genetic Code? The genetic code is simply a set of rules in which information ciphered in RNA or DNA sequences are converted into proteins (amino acid) by living cells
  3. What are Amino Acids? Amino acids are the basic building blocks of all proteins.
  4. What is a Nucleic Acid? Nucleic acids are basically large molecules where genetic information is stored. The nucleic acids are made of nucleotides. Each nucleotide is made of a nitrogenous base, sugar with five carbon atoms and a phosphate group.
  5. What are the two types of Nucleic Acids? There are two types of nucleic acids:
    - Deoxyribonucleic acid AKA DNA
    - Ribonucleic acid AKA RNA
  6. What are Chromosomes? Where are they located? Chromosomes of a cell are located in the cell nucleus. They hold genetic information and are made of DNA.
  7. What is DNA?
    DNA is a nucleic acid that has specific genetic information for the development and function of ALL living things. It has two strands. Each strand is coded with a four-letter alphabet: A (adenine), T (thymine), C (cytosine), and G (guanine). These letters form complementary base-pairs: A only bonds with T, C only bonds with G. The three main functions of DNA are;
    - The long term storage of info
    - Holding the sequence of bases which will be interpreted and deciphered into RNA
    - Encode the sequence of amino acid to proteins, using the genetic code
  8. What is RNA?
    RNA is a much more difficult concept compared to DNA. Along with DNA and proteins, RNA is one of the three major biological macromolecules that are essential to all living things. Note: The flow of genetic information to a cell is from DNA to RNA to Proteins (Proteins are known as the “workhorses of a cell”). RNA is a nucleic acid and made of nucleotides. It is single-stranded and not double-stranded like DNA. In RNA, A bonds with U instead of T. One of RNA’s function is copying DNA’s code. It bonds to a strand of DNA by creating a sequence of the code’s complementary base pairs.
  9. What is Guided RNA?
    The real beauty of RNA is the complexity of it and the wide horizon of roles it actually has. One important type of RNA (especially when it comes to gene editing) is guide RNA. Guide RNA can locate any part of the DNA. This type of RNA can be programmed (which means that if scientists can identify what a particular DNA sequence looks like, they can program the gRNA to find that segment of DNA (target DNA) in any cell.

What is CRISPR-Cas9?

For the longest time, the vast majority of biomedical interventions have attempted to enhance humans and restore something that is viewed to be deficient, such as vision and hearing impairment. However, now, thanks to scientific development in a wide array of fields, we are so close to completing our mission to achieve these ambitions.

The discovery of CRISPR-Cas9 as a gene-editing method was revolutionary.

Up until recently, CRISPR-Cas9 has been the main technology used when it comes to gene editing. When this system was first introduced, the whole world was in awe as they had never seen anything like it. Before CRISPR, gene editing was basically the idea of manipulating genetic information in blocks and big pieces. Think of it like editing a book by only being able to rip out the pages — and not the real words.

There are two distinct parts when it comes to CRISPR-Cas9 — the guide RNA and the Cas9 Protein. As we already know, the guide RNA is a type of RNA that can navigate any part of the DNA. When scientists have successfully designed the gRNA to find and combine a specific sequence in the DNA, they insert it into an endonuclease (an enzyme that breaks down a nucleotide chain into two or more shorter chains) called the Cas-9 protein. After, the guide RNA directs the Cas-9 to the target DNA. As soon as the Guide RNA and Cas-9 reach the target DNA, the Cas-9 protein cuts the target DNA at a specific part called the PAM (think of this section of the DNA as those cutting lines on the labels of shirts that are obviously intended to be cut). After the Cas-9 makes the cut, the DNA attempts to repair itself by bonding random base-pairs together. However, before allowing the DNA to do that, scientists quickly add DNA primers that act as a blueprint for which base-pairs will bond in order to make the edit the scientists want!

The process of CRISPR-Cas 9

Limitations of CRISPR

Now, there is no question that CRISPR Cas9 is an amazing system. However, unfortunately, there are limitations.

  1. Efficiency

To begin with, CRISPRs efficiency varies in a huge way. On average, depending on the case, CRISPR Cas9’s efficiency could range from 50% to 80%. This is a horrible range for gene editing as there is no consistency and is surely not worth the risk.

Many different organizations have considered using Cas 9 on reproductive cells such as eggs and sperm. If changes are made to these cells, the genes could be passed down to future generations in the subject’s family. This could be extremely detrimental to the family due to the low-efficiency rate. Hence, if the subject was to have a complication during the process, we could potentially be giving a whole family (from generation to generation) a bad gene (that is the root cause of a genetic disorder).

2. Cuts in Unintended Areas

CRISPR Cas9 has also been known to cut the DNA molecules at places other than the PAM. This is extremely bad as it can introduce a negative mutation.

3. Not a Clean Cut

In addition to cutting in the wrong place, CRISPR has been known to avoid making clean-cuts which will cause serious genetic damage.

What is Prime Editing?

As you can see, although CRISPR Cas 9 is pretty awesome, it still has many problems. So, the question is: Is there a system similar to CRISPR Cas 9 without the unnecessary risks?

Introducing *drum roll please* the revolutionary gene-editing system known as Prime Editing! Prime editing is a rather new method discovered by a research group called the “Liu Group.” This system allows us to edit and replace specific parts of DNA.

Search-and-Replace Genome Editing System

What makes Prime Editing so much better than CRISPR is the fact that it eliminates the possibility of off-target mutations and unintended editing.

How does Prime Editing work?

1. The prime editor uses a Cas9 enzyme (modified to only cut one strand of DNA), and a reverse transcriptase enzyme, which has the ability to generate completely new DNA by copying an RNA. The first step involves an engineered Prime Editing Guide RNA (pegRNA) that shows the editor how to get to the target DNA in order to cut it.

2. The pegRNA has two distinct parts — A section that binds to the cut DNA, and a section of RNA letters that decipher the wanted edit. When pegRNA binds to the DNA, this prepares the strand to have completely new DNA with the four-letter alphabet.

3. Now, in order to transfer the edited sequence from the pegRNA to the target DNA, the reverse transcriptase reads the RNA and attaches the corresponding alphabet (A (adenine), T (thymine), C (cytosine), and G (guanine)) to the end of the cut DNA.

4. Then, an endonuclease naturally removes the old part of DNA and covers the genome with the new letters.

5. Now, as you can see, the Target DNA is left with one edited strand and one unedited strand. Therefore, to resolve this, a different guide RNA shows the prime editor where exactly to cut the unedited strand.

6. After this cut, the cell is prompted to repair the cut strand using the edited strand as an instruction manual.

Importance of Prime Editing

Examples of Genetic Disorderslocated on each Chromosome

According to a study published in 2017, 1 in 5 people are born with some kind of genetic disease and many of these diseases are in fact inherited. If a cure for these genetic disorders is not found, the percentage of the population with these disorders will grow at an unbelievable rate. Therefore, in order to cure most of these genetic disorders, we have to reprogram mutated DNA — making Prime Editing so important. Prime Editing is truly revolutionary and has the ability to correct 89 percent of genetic deficiencies.

Ethics of Prime Editing

Now, the emerging of CRISPR and Prime Editing have raised many ethical questions and concerns. Below I will address some of these concerns along with a quick description.

1. Inequality Based On Wealth
With the emerging of Prime Editing, economic inequality based on wealth will be extremely clear due to the increase of gene editing around the world. Naturally, the cost of many procedures will be different. However, if there is one thing that is certain, it is that almost all of these procedures will cost a great sum of money. For instance, it will cost anywhere from $15,000 to $25,000 to select the gender of your future baby. This will be a clear example of inequality as most people cannot afford to pay this much money. In addition to the cost, gene editing can also result in a baby being brought in this world with the genes needed to succeed in life, primarily due to physical characteristics. For example, according to a number of studies, people with greater height have been known to be more successful in life. Another example includes the change of gender, women have been known to have a salary that is 12% less compared to the salary of a man.

2. Potential for Eugenics
Eugenics is the overall concept of improving the human species by basically selecting traits that go against genetic and physical differences. The problem with EUgenics is that you could potentially be allowing disparities to exist in this world. For instance, some may say that people could use gene editing to create the “perfect human being” with the perfect race, eyes, length, gender, etc. (whatever these may be).

3. Possible Confusion Over Government’s Role
Another controversial topic is the idea of the government being able to decide how parents deal with their children's DNA. Some may say that the use of gene editing should be regulated by the government. However, if gene editing is, in fact, safe, then why should the government be able to decide the important questions like:1. How people are allowed to use this tool? 2. What type of people can use this tool?

4. Religion

Christianity — In the bible, it states that “any scientific or technological advance should be used for the improvement of humanity.” However, some may say that gene editing being used to reverse diseases “would parallel the role of God” and in other words, be “Cheating God.” In addition, in the bible, it also expresses “the importance of the relationship between the baby and the mother in the womb.” So the real question is: would the use of these genetic engineering systems interfere with the development of the soul?

Judaism
Assuming that genetic engineering does in fact work, many believe that the whole concept would, in fact, be accepted in Judaism. This is because, in Judaism, people believe that “human life has an infinite value” and that “he who saves one life; has saved the whole world.”

Islam
Although there are many things in Islam that support the idea of genetic engineering, there are still a few things in the Quran that would say otherwise. For instance, in the Quran, it states “Believers, fortify yourselves with patience and prayer. God is with those that are patient.” Therefore, due to the fact that humans must wait to overcome suffering, there might be a huge controversy regarding the topic in Islam.

Finally, to end it off:

The advance of genetic engineering makes it quite conceivable that we will begin to design our own evolutionary progress.

- Isaac Asimov

--

--