The Next Step in Developing Preventive and Curative Gene Therapies

Diseases such as cystic fibrosis, Huntington’s disease, Tay-Sachs disease, sickle cell disease, and several types of cancer are hereditary, meaning they can be determined by genetic factors passed on from parents to children.

These diseases severely impact the quality of life of not only the people suffering from them, but their families, friends, and caretakers as well.

Due to the genetic nature of these diseases, certain people have an increased likelihood of getting them. This raises concerns for those with a family history of these diseases and questions of if they should be tested for biomarkers and/or take preventative actions.

Gene Editing

Gene editing is a technology that enables scientists to make changes to DNA which could lead to changes in physical traits, such as eye and hair colour, as well as, risk of disease.

It is performed using enzymes, particularly nucleases, engineered to target a specific DNA sequence, introduce cuts into the strands, enable the removal of existing DNA and the insertion of replacement DNA.

Scientists are currently developing gene therapies, which involve gene editing, to prevent and treat diseases in humans. There are two different categories of gene therapies: germline therapy and somatic therapy.

Germline therapies change DNA in reproductive cells (i.e. sperm and eggs) so any changes to the DNA will be passed down from generation to generation, while somatic therapies target non-reproductive cells, so changes made in these cells will affect only the person who receives the gene therapy.

Deoxyribonucleic Acid (DNA) 🧬

DNA stores information in a code that is made up of four chemical bases: adenine (A), guanine (G), cytosine (C ), and thymine (T).

These bases pair up with each other, A with T and C with G, to form base pairs. The order of these bases determines the information for building and maintaining an organism. Each of the bases is also attached to a sugar and a phosphate molecule. Together, a base, sugar, and phosphate molecule are called a nucleotide.

Nucleotides are arranged in two long strands that form a spiral called a double helix. The double helix is structured similarly to a ladder where the sugar and phosphate molecules form the vertical sidepieces and the base pairs form the rungs.

CRISPR-Cas9

Short for Clustered Regularly Interspaced Short Palindromic Repeats

CRISPR-Cas9 is a well-known genome editor that uses an enzyme called Cas9 which navigates to its target DNA guided by an RNA molecule. It then edits/modifies the DNA, which can deactivate genes, or inserts a desired sequence instead.

At precise locations in the genome where changes are wanted, CRISPR creates double-strand breaks (DSBs). However, CRISPR does also rely on cells’ natural repair processes, which is not ideal, because as the cell is repairing itself, there could be damage or unwanted mutations created.

But what can we do to avoid these mutations?

Enter..

Prime Editing

Prime editing is a new and improved gene editing technique that can rewrite DNA by adding, deleting, or replacing base pairs by cutting only a single strand of DNA (modified Cas9 enzyme) with increased precision and efficiency, resulting in fewer byproducts or unwanted modifications.

David R. Liu, a biochemist at Broad Institute of MIT and Harvard, and his Research Group have developed this versatile way to edit genomes with the ability to make larger edits than previously possible (up to 44 base pairs for insertion and up to 80 base pairs for deletion).

The Process

Prime editing can be thought of as a ‘search and replace’ gene editing technique because it searches for the mutated gene in an organism, and then replaces it using insertions, deletions, or substitutions of the desired genetic changes.

“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.” — David Liu

The prime editor complex includes a modified Cas9 enzyme and a reverse transcriptase enzyme, which can generate new DNA by copying an RNA template. An engineered prime editing guide RNA (pegRNA) then sends the editor to its target, where Cas9 cuts the DNA.

The pegRNA has two special components:

  • a section that binds to the DNA that’s been cut and prepares it to have new letters added
  • a section of RNA letters that encodes the desired edit

The reverse transcriptase then reads the RNA and attaches the corresponding DNA letters to the end that has been cut. An endonuclease in the cell naturally removes the old segment of DNA and seals the new letters into the genome. Now, the target site is left with one edited strand and one unedited strand.

A different guide RNA then directs the prime editor to cut the edited strand to solve the mismatch. This cut prompts the cell to remake that strand, using the edited strand as a template, and completes the edit.

Accuracy

Prime editing is more complex and precise than CRISPR. While CRISPR relies on only one pairing step (the guide RNA must pair with the target DNA), prime editing requires three separate steps which Liu says he believes might be the secret to its accuracy.

“If any one of those three DNA pairing events fail, then prime editing can’t proceed,” says Liu. “We believe that those three independent pairing events each provide an opportunity to reject off-target sequences,” he adds.

The three main components include the Cas9 enzyme, the reverse transcriptase enzyme, and the guide RNA. The primer, a part of the guide RNA, must bind to the target site while the newly introduced DNA must bind to the original site.

This minimizes the damage in the genome resulting in a more efficient healing process than that of CRISPR. Prime editing can insert, delete, or modify individual DNA letters but also a sequence of multiple letters into a genome with minimal damage to DNA strands.

Importance

Prime editing allows researchers to edit more types of genetic mutations than before. Gene editing is a huge advancement to curing genetic disorders that are affecting millions around the world.

Genetic disorders can cause such severe health problems even before birth. In severe cases, these conditions may cause a miscarriage of an affected embryo or fetus. Affected infants could also pass away before or soon after birth.

Unfortunately, very few treatments are available for these severe genetic conditions and health professionals can only provide supportive care, such as pain relief or breathing assistance.

The future

Prime editing is more advanced than other genome engineering softwares such as ZFNs or TALENs. It presents tools for biological advancements, the development of treatments for diseases, and the modification of plants for more resistant or higher yielding crops.

Imagine the ability to stop cancer or hereditary diseases, like breast cancer, from happening simply by editing out the genes that cause it. With prime editing, millions of people worldwide will no longer have to endure long, draining, and painful treatment processes.

Editing genes to prevent genetic disorders and/or hereditary diseases could have an enormous impact on reducing the costs of healthcare and long term disability care to revolutionize methods of medical treatment.

Imagine our world…but minus the genetic diseases.

Maybe we won’t have to imagine much longer.

Say ‘👋🏻’ to a new and improved world. 🌍

TL;DR

Many diseases such as cystic fibrosis, Huntington’s disease, Tay-Sachs disease, sickle cell disease, and several types of cancer are hereditary.

Gene editing is a technology that enables scientists to make changes to DNA which could lead to changes in physical traits, as well as, risk of disease.

CRISPR-Cas9 is a well-known genome editor that uses an enzyme called Cas9 to navigate to its target DNA , edit/modify it, or insert a desired sequence instead.

However, CRISPR creates double-strand breaks (DSBs) and relies on cells’ natural repair processes which can result in damage or unwanted mutations.

Prime editing is a new and improved gene editing technique that can rewrite DNA while cutting only a single strand of DNA. It is precise, efficient, and results in fewer byproducts or unwanted modifications.

Prime editing can be thought of as a ‘search and replace’ gene editing technique because it searches for the mutated gene in an organism, and then replaces it using insertions, deletions, or substitutions of the desired genetic changes.

Prime editing is more complex and precise than CRISPR. While CRISPR relies on only one pairing step, prime editing requires three separate steps, increasing its accuracy.

Editing genes to prevent genetic disorders and/or hereditary diseases could have an enormous impact on reducing the costs of healthcare and long term disability care to revolutionize methods of medical treatment.

Thank you for reading! If you enjoyed this article, feel free to share it with your friends and family and leave a 👏! Be sure to also connect with me on LinkedIn!

BCI Researcher and Innovator at TKS

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