Mitochondrial gene editing
Mitochondrial base editing is a technique to change the mitochondrial DNA, which can be used to create animal disease models or potentially, and more importantly, to cure patients. This article will talk about what mitochondria and mitochondrial disease are, what gene editing is, why genetic engineering is so difficult in mitochondria and how base editing is such an important new technique. Furthermore, this article will explain how next generation sequencing is used to measure the efficiency and even more importantly, to measure potential devastating off-target effects.
What are mitochondria?
Mitochondria are small particles in a cell. They have their own DNA, the mitochondrial DNA, that is responsible for thirteen proteins in the mitochondria. However, for mitochondria to function, they also need hundreds of proteins that have their genetic information on one of the chromosomes in the nucleus.
What are mitochondrial diseases?
Mitochondrial diseases are a group of genetic disorders that are characterized by defects in mitochondrial function. They can be caused by mutations in the mitochondrial DNA or, more common, mutations in the DNA of the nucleus. Each cell has a set of each chromosome in the nucleus, but each cell has hundreds to thousands of mitochondrial DNA copies. And even in a healthy person, some of these copies might have a mutation. But in a healthy person, the majority of DNA copies does not have mutations. In patients, a high percentage of mitochondrial DNA copies has a mutation. This percentage is called the heteroplasmy level.
What is gene editing?
The first attempts to change the DNA used chemicals or radiation, but there was no way of controlling where the changes would happen with these methods. In the 1970s and 1980s the first attempts were made to specifically target the changes by a method called homologous recombination. This method is very precise but quite inefficient and time-consuming.
Today, there are several gene-editing techniques: CRISPR-Cas9, Zinc Finger Nucleases (ZFN), TALENs, restriction endonucleases and the recently developed technique DdCBE.
In this article you will learn more about the most famous one, CRISPR-Cas9, and why this doesn’t work to edit mitochondrial DNA, and on DdCBE, which is creating new possibilities to modifiy the mitochondrial DNA.
CRISPR-Cas9
CRISPR-Cas9 is the best-known gene editing technique. That’s mostly because the development of the CRISPR-Cas9 editing technique caused a revolution in gene editing possibilities, due to its cheapness and simplicity. The invention of this technique was considered so important that Emmanuelle Charpentier and Jennifer Doudna won the Nobel prize in Chemistry for this.
How does CRISPR-Cas9 gene editing work? Cas9 is an enzyme that works as a pair of scissors that can cut the DNA. But to know where to cut the DNA exactly, it needs a piece of RNA, called guide RNA. This guide RNA is specifically designed to find and bind to a specific sequence in the DNA to “guide” Cas9 where to cut the DNA. The cell will recognize the DNA damage and will try to repair it. Sometimes the cell can repair it perfectly, but sometimes it fails and makes a mistake. This is what we want! The gene editing is actually the result of a mistake during the repair of the cut.
CRISPR-Cas9 can change the chromosomal DNA, but the guide RNA can’t reach the mitochondrial DNA, so it can’t be used to study or cure mitochondrial diseases.
DdCBE
About two years ago, another technique was discovered, DdCBE (double-stranded DNA deaminase-derived cytosine base editor). In contrast with the other techniques, this one doesn’t cut the DNA, but really changes it. Unfortunately, it can only change certain positions, not everything, but it’s an important step forward towards efficient gene editing. It is a fast and relatively easy technique that can also reach the mitochondrial DNA, in contrast with CRISPR-Cas9.
Can mitochondrial gene editing be used as therapy?
Mitochondrial gene editing therapy involves modifying the DNA of the mitochondria to correct or replace the faulty genes that cause mitochondrial disease. As mentioned higher, CRISPR/Cas9 can’t be used, but DdCBEs are more promising. However, mitochondrial gene editing therapy is still in the experimental stage and there are many challenges to overcome, including ensuring the safety and efficacy of the treatment and addressing ethical concerns surrounding genetic engineering. Further research is needed to determine the potential benefits and risks of mitochondrial gene editing therapy and to develop safe and effective treatment options for mitochondrial diseases.
How can NGS used to study (mitochondrial) gene editing?
Obviously, when trying to modify the DNA, it is necessary to check whether it worked and the DNA has changed. NGS sequencing is the perfect method for this, especially for mitochondrial DNA, as there are many copies in each cell. Amplicon-resequencing can be used to check a small region of the DNA, surrounding the target-site.
All types of gene editing techniques are relatively precise in where they cut the DNA, but not 100%! They make mistakes and sometimes cut the wrong piece of DNA (off-target). So, while they might correct the genetic disease in the cell/body, they could at the same time create another disease. So, it is crucial to carefully check the whole genome for off-target effects and NGS is the easiest technique to do this.
Here you can find some examples of off-target analysis we did. Do you want us to analyse your NGS data or do you have questions, just contact us.