New tool uses an interbacterial toxin to achieve precise mitochondrial gene editing without CRISPR side-effects.

Mitochondria – often called the “powerhouse” of the cell – contain their own DNA (mtDNA). Just like nuclear DNA, it is subject to mutations which can result in diseases such as diabetes, or predispositions to conditions like Alzheimer’s disease and Parkinson’s disease [1]. What can be done about these mutations?

Longevity.Technology: Mitochondrial mutations can lead to a variety of diseases and conditions, many of which are both poorly-understood and have life-limiting consequences. Although this new technology has a long way to go before we are using it to tinker with our own mitochondria, the benefits of using it in the lab to properly understand, mitigate and hopefully even prevent a range of devastating diseases would have tremendous benefits for extended lifespan and healthspan for patients.

The explosion in research and interest in therapeutic gene editing is evident; just last month, Guangping Gao, PhD, President of the American Society of Gene and Cell Therapy and a gene therapy researcher at the University of Massachusetts Medical School said “Gene therapy is now in its golden age,” as genome editors “open even more avenues for treating disease [2].”

Using RNA and the Cas9 enzyme, pieces of genetic material can be added or deleted, changes to the DNA can be made by replacing an existing segment with a customised DNA sequence. However, double-strand breaks in DNA are dangerous and can cause translocations of DNA, a complicated mix of  harmful products and suppress cells’ cancer-fighting ability. Prime Editing has improved the process, but now a team from Howard Hughes Medical Institute (HHMI) has looked at how problems in mitochondrial DNA can be tackled.

The structure of mitochondria make it difficult for RNA to enter in order to get to mtDNA and they lack efficient mechanisms for repairing double-strand breaks. These obstacles mean CRISPR is not the tool to use for mtDNA editing. The HHMI team has circumvented these obstacles with a toxin that bacteria use to wage warfare on each other and the method devised is, according to Joseph Mougous, an HHMI Investigator at the University of Washington, “the first precision gene editor for mitochondrial DNA [3]”.

The interbacterial toxins are called bacterial deaminases; they can cause genetic mutations by removing nitrogen-containing parts of DNA and RNA bases and converting the base cytosine to uracil. Lots of deaminases work on single strands of DNA, RNA and free nucleotides or nucleosides. However, the HHMI team found one that worked on double-stranded DNA, the now aptly-named double-stranded DNA deanimase toxin A (DddA).

The team bound DddA to TALENs (a pre-CRISPR gene editor that uses transcription activator-like effector nucleases (TALEN), restriction enzymes that can be engineered to cut specific sequences of DNA) and added UGI (Uracil Glycosylase Inhibitor) which prevents uracil (a RNA base) from being cut out and replaced with cytosine. This created a CRISPR-free mitochondrial base-editing tool. The team used 3D imaging data to split the DddA protein into two halves – an N-terminal part and a C-terminal half – to counteract the toxic nature of the deanimase.

DddA proved to be an capable editor, with an efficiency ranging from 4.6% to 49% [4]; the tested the editor by making single-letter edits in five different human mitochondrial genes, finding it edited approximately 20% to 40% of the mitochondria targeted.

“20% to 40% might not sound particularly impressive, but many genetic diseases can be treated by levels of correction that are in that ballpark,” says David Liu, PhD, of Harvard University and the Broad Institute. “You rarely need to correct 100% of genes to have a benefit to a prospective patient [5].”

The team hope that if they can find a deanimase that switch one base for another, there will be other deanimases that will allow them to make other mitochondrial DNA alterations.

Vamsi Mootha, MD, of Massachusetts General Hospital and the Broad Institute said: “This is a transformative technology for my field. It will now be possible to create mouse models of mitochondrial DNA disease — this has been super difficult until now [6].”

[1] https://www.nature.com/scitable/topicpage/mtdna-and-mitochondrial-diseases-903/
[2] https://www.hhmi.org/news/precision-genome-editing-enters-the-modern-era
[3] https://www.hhmi.org/news/how-to-precisely-edit-mitochondrial-dna
[4] https://www.nature.com/articles/s41586-020-2477-4
[5] https://bit.ly/3jwi9nd
[6] https://phys.org/news/2020-07-molecular-tool-precisely-mitochondrial-dna.html