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 discuss what mitochondria and mitochondrial disease are, what gene editing is, why genetic engineering is so tricky in mitochondria and how base editing is such an important new technique. Furthermore, this article will explain how next-generation sequencing measures efficiency and, even more importantly, estimate potential devastating off-target effects.
What are mitochondria?
Mitochondria are tiny particles in a cell. They have their own DNA, the mitochondrial DNA, which is responsible for thirteen proteins in the mitochondria. However, for mitochondria to function, they also need hundreds of proteins whose genetic information is on one of the chromosomes in the nucleus.
What are mitochondrial diseases?
Mitochondrial diseases are a group of genetic disorders characterised by defects in mitochondrial function. They can be caused by mutations in the mitochondrial DNA or, more commonly, mutations in the DNA of the nucleus. Each cell has a set of chromosomes 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. However, most DNA copies do not have mutations in a healthy person. In patients, a high percentage of mitochondrial DNA copies have a mutation. This percentage is called the heteroplasmy level.
What is gene editing?
The first attempts to change DNA used chemicals or radiation, but these methods did not allow for control over the changes. 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 relatively inefficient and time-consuming.
Several gene-editing techniques exist, such as 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 it doesn’t work to edit mitochondrial DNA, as well as about DdCBE, which is creating new possibilities to modify mitochondrial DNA.
CRISPR-Cas9
CRISPR-Cas9 is the best-known gene editing technique. That’s mainly 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 crucial that Emmanuelle Charpentier and Jennifer Doudna won the Nobel Prize in Chemistry for it.
How does CRISPR-Cas9 gene editing work? Cas9 is an enzyme that works like scissors that can cut DNA. However, a piece of RNA called guide RNA must be cut to know where to cut the DNA precisely. 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 recognise the DNA damage and will try to repair it. Sometimes, the cell can fix it perfectly, but sometimes, it fails and makes a mistake. This is what we want! The gene editing results from an error during the repair of the cut.
CRISPR-Cas9 can change chromosomal DNA, but the guide RNA cannot reach mitochondrial DNA, so it cannot 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 methods, this one doesn’t cut the DNA but changes it. Unfortunately, it can only change certain positions, not everything, but it’s an essential step towards efficient gene editing. It is a fast and relatively easy technique that can also reach the mitochondrial DNA, contrasting 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 above, 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 DNA, it is necessary to check whether it worked and whether 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 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 simultaneously create another disease. So, it is crucial to check the whole genome for off-target effects carefully, and NGS is the most straightforward technique.
Here are some examples of off-target analysis we performed. If you want us to analyse your NGS data or have questions, just contact us.