By Saumya Sharma
DddA complex system diagram. Composed of an mtDNA targeting sequence, TALE protein, DddA half, and UGI.
CRISPR has been the leading gene-editing tool in genetics for the past 8 years, but what if there was a new targeted option that addressed issues in the energy systems in the human body? An option that could undo mutations in the mitochondria.
Before delving into the logistics of gene editing, it is important to understand the basics. DNA is the name of a chain of code that dictates every aspect of a living thing. It is housed in the nucleus of the cell and is made up of four main bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Additionally, there are other bases that are used in a different process/type of DNA, the most important of which, for understanding mitochondrial gene editing, being uracil (U), which is used in RNA (a version of genetic material that uses U instead of T). These bases fall into two categories: pyrimidines (C, T, and U) and purines (A and G). Each pyrimidine pairs with a purine. Each set of bases encodes for enzymes, which are proteins in charge of governing cell behavior by catalyzing reactions. Any changes that occur to the order of bases in the DNA are called mutations, which can affect the function and efficacy of the enzymes.
As most teens know, the mitochondria are the “powerhouse of the cell.” In other words, the mitochondria are the main provider of the cell’s energy supply, called ATP (adenosine triphosphate). While the origin of the mitochondria in human cells is not confirmed, the prevailing theory is that the organelle was at one point a single-celled organism that eventually merged with other cells. This is assumed because of the presence of the phospholipid bilayer (a thin, double-layered membrane that usually separates the outside of a cell from the inside) that encases the mitochondria, and the presence of a separate set of DNA called mtDNA. The mtDNA encodes 13 proteins that are involved in ATP production. If mutations occur in the mtDNA, there would be direct effects on the wellbeing of the mitochondria, thus affecting the rest of the cell as well. This is why scientists are working on developing a tool to reverse the mutations in mtDNA.
The solution came in the form of a cytidine deaminase enzyme called DddA. Cytidine deaminase enzymes govern the fixing/replacing of pyrimidine bases in DNA. DddA works by targeting DNA and facilitating the change between cytosine and uracil.
Now, for DddA to work, there are a few obstacles that scientists had to eliminate. The first, and arguably the most critical, was the toxicity of DddA. DddA is normally toxic to mammalian cells. Scientists were able to bypass this obstacle by splitting DddA into two halves and binding each half to a separate TALE (Transcription activator-like effector) protein (see image above). This way, when the DddA halves needed to be together on the mtDNA, they could be connected easily while still being separated to counter the protein’s toxicity.
The next challenge was getting the DddA parts into the mitochondria. Because of the phospholipid bilayer around the mitochondria, scientists had to use a sequence of amino acids to allow the DddA protein to pass through the phospholipid bilayer, and also target the part of the mtDNA that needed to be edited.
Furthermore, an issue arose with the fact that DddA replaces each C with a U in the selected mtDNA. As stated earlier, U is only used in RNA, not DNA. DNA correction proteins, uracil DNA glycosylase (UNG), actively work in the cell to ensure that all DNA and mtDNA have the correct bases. Since U is an RNA base, if the UNG finds this base, it will try to undo the work the scientists did. To prevent this from happening, scientists added a UNG inhibitor to the DddA complex. This inhibitor blocked UNG from changing the U back to a C. By blocking this change, the cell auto-adjusted the mtDNA with the correction.
After all of these changes, the original DddA is cut in half, attached to a UNG inhibitor, and a TALE protein. This final complex is called a DddA-derived cytosine base editor (DdCBE).
DdCBE is efficient at correcting mtDNA mutations for a few reasons. Firstly, it works on double-stranded mtDNA; instead of having to unravel and separate the mtDNA sections, DdCBE is able to work faster with less room for error. Secondly, it does not have to cut the mtDNA strands to access the bases. Because the mitochondria do not code for as many proteins as the nucleus of the cell, there are fewer DNA damage-repair systems in place. By not slicing the DNA strands, DdCBE is able to reduce time and potential error. Finally, DdCBE uses an amino acid sequence to pass into the mitochondria and target the mtDNA.
This new mitochondrial DNA editing tool is vital in helping individuals with mitochondrial energy issues that stem from their genetics. On top of this, it provides a revolutionary new perspective in the world of gene editing and genetic engineering.
Special thanks to Beverly Mok for access to the journal article and research.
Doudna, J. A., & Sternberg, S. H. (2018). A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution (Illustrated ed.). Mariner Books.
Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. V., Raguram, A., Hsu, F., Radey, M. C., Peterson, S. B., Mootha, V. K., Mougous, J. D., & Liu, D. R. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 583(7817), 631–637. https://doi.org/10.1038/s41586-020-2477-4