Bacterial Toxin Gene Editor Precisely Edits Mitochondrial DNA
Bacterial Toxin Gene Editor Precisely Edits Mitochondrial DNA
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The genome in mitochondria – the cell’s energy-producing organelles – is involved in disease and key biological functions, and the ability to precisely alter this DNA would allow scientists to learn more about the effects of these genes and mutations. But the precision editing technologies that have revolutionized DNA editing in the cell nucleus have been unable to reach the mitochondrial genome.
Now, a team at the Broad Institute of MIT and Harvard and the University of Washington School of Medicine has broken this barrier with a new type of molecular editor that can make precise C•G-to-T•A nucleotide changes in mitochondrial DNA. The editor, engineered from a bacterial toxin, enables modeling of disease-associated mitochondrial DNA mutations, opening the door to a better understanding of genetic changes associated with cancer, aging, and more.
The work is described in Nature, with co-first authors Beverly Mok, a graduate student from the Broad Institute and Harvard University, and Marcos de Moraes, a postdoctoral fellow at the University of Washington (UW).
The work was jointly supervised by Joseph Mougous, UW professor of microbiology and an investigator at the Howard Hughes Medical Institute (HHMI), and David Liu, the Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, professor of chemistry and chemical biology at Harvard University, and HHMI investigator.
“The team has developed a new way of manipulating DNA and used it to precisely edit the human mitochondrial genome for the first time, to our knowledge – providing a solution to a long-standing challenge in molecular biology,” said Liu. “The work is a testament to collaboration in basic and applied research, and may have further applications beyond mitochondrial biology.”
Agent of bacterial warfare
Most current approaches to studying specific variations in mitochondrial DNA involve using patient-derived cells, or a small number of animal models, in which mutations have occurred by chance. “But these methods pose major limitations, and creating new, defined models has been impossible,” said co-author Vamsi Mootha, institute member and co-director of the Metabolism Program at Broad. Mootha is also an HHMI investigator and professor of medicine at Massachusetts General Hospital.
While CRISPR-based technologies can rapidly and precisely edit DNA in the cell nucleus, greatly facilitating model creation for many diseases, these tools haven’t been able to edit mitochondrial DNA because they rely on a guide RNA to target a location in the genome. The mitochondrial membrane allows proteins to enter the organelle, but is not known to have accessible pathways for transporting RNA.
One piece of a potential solution arose when the Mougous lab identified a toxic protein made by the pathogen Burkholderia cenocepacia. This protein can kill other bacteria by directly changing cytosine (C) to uracil (U) in double-stranded DNA.
“What is special about this protein, and what suggested to us that it might have unique editing applications, is its ability to target double-stranded DNA. All previously described deaminases that target DNA work only on the single-stranded form, which limits how they can be used as genome editors,” said Mougous. His team determined the structure and biochemical characteristics of the toxin, called DddA.
“We realized that the properties of this 'bacterial warfare agent' could allow it to be paired with a non-CRISPR-based DNA-targeting system, raising the possibility of making base editors that do not rely on CRISPR or on guide RNAs,” explained Liu. “It could enable us to finally perform precision genome editing in one of the last corners of biology that has remained untouchable by such technology – mitochondrial DNA.”
“Taming the beast”
The team's first major challenge was to eliminate the toxicity of the bacterial agent – what Liu described to Mougous as “taming the beast” – so that it could edit DNA without damaging the cell. The researchers divided the protein into two inactive halves that could edit DNA only when they combined.
The researchers tethered the two halves of the tamed bacterial toxin to TALE DNA-binding proteins, which can locate and bind a target DNA sequence in both the nucleus and mitochondria without the use of a guide RNA. When these pieces bind DNA next to each other, the complex reassembles into its active form, and converts C to U at that location – ultimately resulting in a C•G-to-T•A base edit. The researchers called their tool a DddA-derived cytosine base editor (DdCBE).
The team tested DdCBE on five genes in the mitochondrial genome in human cells and found that DdCBE installed precise base edits in up to 50 percent of the mitochondrial DNA. They focused on the gene ND4, which encodes a subunit of the mitochondrial enzyme complex I, for further characterization. Mootha's lab analyzed the mitochondrial physiology and chemistry of the edited cells and showed that the changes affected mitochondria as intended.
“This is the first time in my career that we’ve been able to engineer a precise edit in mitochondrial DNA,” said Mootha. “It's a quantum leap forward – if we can make targeted mutations, we can develop models to study disease-associated variants, determine what role they actually play in disease, and screen the effects of drugs on the pathways involved.”
One goal for the field now will be to develop editors that can precisely make other types of genetic changes in mitochondrial DNA.
“A mitochondrial genome editor has the long-term potential to be developed into a therapeutic to treat mitochondrial-derived diseases, and it has more immediate value as a tool that scientists can use to better model mitochondrial diseases and explore fundamental questions pertaining to mitochondrial biology and genetics,” Mougous said.
The team added that some features of DdCBE, such as its lack of RNA, may also be attractive for other gene-editing applications beyond the mitochondria.
Mok et al. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature. DOI: https://doi.org/10.1038/s41586-020-2477-4
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