by Yazmin I. Rovira Gonzalez, Ph.D., JHU Alumna
CRISPR-Cas9 technology is a powerful tool for editing genomes, as it allows researchers to alter DNA sequences and modify gene function. It can potentially correct genetic defects, treat, and prevent the spread of diseases. CRISPR works alongside different types of “Cas” proteins found in bacteria, where they usually help defend against viruses. The Cas9 protein is the most used by scientists, and it can be programmed to find and bind almost any desired target sequence, only by providing it with a piece of RNA to guide it in its search (guide RNA).
The CRISPR system discovered, particularly the CRISPR/Cas9 system, editing mitochondria, a membrane-bound structure found in the cytoplasm of eukaryotic cells, where cellular respiration and (most) energy produced in a cell, is challenging to accomplish because of the problems involved in guide RNA import. Mitochondria have specific biological properties that make it difficult for genetic manipulation. Specifically, they have unique pathways used to express and import mitochondrial proteins, multiple copies of mitochondria, and mtDNA. There is a risk of cell injury or apoptosis caused by targeting mitochondrial DNA for gene editing.
On July 8th, 2020, microbiologist Joseph Mougous and colleagues at the University of Washington in Seattle published an article in Nature where they describe how a peculiar enzyme enabled them to edit the genomes of mitochondria. Initially, Mougous and colleagues wanted to understand how bacteria arrange toxins to combat one another, and whether this had any impact on bacterial ecosystems. To “follow” these toxin-producing bacteria, they needed a toxin that could leave a trace, so they looked for one that can change the DNA of the organisms the toxin attacks.Ultimately, they chose to follow a toxin made by the bacterium Burkholderia cenocepacia, which they called DddA.
They found that whenever the toxin encountered the DNA base C, it converted it to a U. Since U is not commonly found in DNA, the cell’s replication machinery reads it as a T, efficiently turning the original DNA base C into a T. Mougous hypothesized that tracing these C-to-T changes would be a sign that the toxin had shaped the bacterial ecosystem. However, what the researchers found was that DddA modified double-stranded DNA, whereas most enzymes of its kind modify only single-stranded DNA. With a few tweaks, they discovered that DddA could be used to edit the double-stranded mtDNA.
Interestingly, DddA converts C to U without inducing any double-strand DNA breaks with typical efficiencies ranging between 5% and 50%, making it well suited to edit the mitochondrial genome since mtDNA lacks efficient mechanisms for repairing double-strand DNA breaks. The ability of DddA to make C-to-T changes was too promiscuous, as it would mutate every C it came across if set loose. To prevent this, the scientists split the enzyme into two pieces that would make the C-to-T changes only when brought together in the right orientation. To control which DNA sequence the enzyme modified, the team then linked each half of the split DddA to proteins that engineered to bind to specific sites in the genome.
More studies are needed to understand better the off-target DNA changes that occur with DddA, particularly the biology behind the efficiency and specificity of this enzyme. In vitro and in vivo strategies to deliver this editing system will be essential for exploring the therapeutic potential in other cell types and animal models of mitochondrial diseases. Regardless, discoveries like this one are important because mutations in mitochondrial DNA can result in devastating disorders, such as Leber’s hereditary optic neuropathy, which causes vision loss and for which there is no proven treatment. Eventually, mitochondrial editing could potentially correct mitochondrial mutations, enabling new gene therapy methods for mitochondrial DNA diseases.
Photo source: https://biomedicalodyssey.blogs.hopkinsmedicine.org/2015/06/johns-hopkins-tackles-genome-editing-with-crisprcas9-symposium/