Author: Anitta Rose Chacko
Technologies such as CRISPR have revolutionised precision genome editing and paved the way for treatment of previously incurable diseases and the generation of accurate disease models. Remarkably, a new study conducted by Mok et al., recently published in Nature, has described a bacterial toxin with the ability to perform precise editing in the previously ‘untouchable’ mitochondrial DNA (mtDNA) genome.
Mitochondria, the cell’s energy producing organelles, have a significant role in biological functions as well a key role in disease, so a method to precisely edit the mtDNA genome has been a long sought-after goal. Editing technologies based on CRISPR have had little success with mtDNA due to the inability to transport guide RNA, a major component of CRISPR-Cas9 technology, into the mitochondria. The new, potentially breakthrough molecular tool which has been engineered by researchers from the Broad Institute of MIT, Harvard and the University of Washington School of Medicine, has been named as RNA-free DddA-derived cytosine base editors (DdCBEs). The research by Mok et al., 2020, enables a precise C•G-to-T•A base conversion to be made without utilising guide RNA, providing a technical advance and the opportunity to better understand mitochondrial disease.
The collaboration which led to the discovery of the new editing tool began with Marcos de Moraes, a postdoc working in the Joseph Mougous led lab at the University of Washington. Moraes recognised the ability of the bacterium Burkholderia cenocepacia to secrete toxin DddA, a cytidine deaminase that catalyses the conversion of nucleotide base cytosine into uracil in neighbouring bacteria to kill them in scarce conditions. Interestingly, Moraes found that the bacterial toxin generates the base conversion in both strands of DNA, which had not yet been identified with previously studied cytidine deaminases. Curious about this discovery, Mougous and his team delved deeper into the bacterial toxin to determine its biochemical characteristics and structure before approaching David Liu, a Howard Hughes Medical Institute (HHMI) investigator, who has previous experience with incorporating cytidine deaminases for use in base editing technology for single-stranded DNA.
Following the structural determination of DddA, the deaminase was divided into two inactive halves, one half containing the C terminus and the other half containing the N terminus of DddA, to overcome the major challenge of toxicity associated with the deaminase. The two inactive halves, ‘Split-DddAtox halves’ were also separately tethered to TALE proteins and tagged with a mitochondria targeting amino acid sequence. The tethering of Split-DddAtox halves to TALE binding proteins enabled the cytidine deaminase to locate and target specific mtDNA sequences and so render the Split-DddAtox halves active as they reassembled and combined, allowing the cytosine to uracil base conversion to occur. Conversely, the addition of the mitochondrial targeting sequence permitted the construct to pass mitochondrial outer and inner membranes without the need for guide RNA.
A further challenge that was presented with the bacterial toxin was that mitochondrial DNA would be converted to uracil as opposed to the DNA specific thymine base. To approach this problem, Mok et al. incorporated a uracil glycosylase inhibitor to each Split-DddAtox half, to protect the base from being replaced with cytosine by the uracil-DNA glycosylase enzyme. This enabled the precise U•G-to-T•A base conversion during the subsequent round of DNA replication as the guanine base on the original complementary strand is replaced by adenine to pair with thymine.
There are several reasons that set this new editing technology apart from previous research on mtDNA genome editing. Primarily, this new molecular tool does not require ‘traditional’ CRISPR RNA guides, exploiting existing transport machinery to transfer protein into mitochondria. Alternative editing approaches, such as TALEN technology, rely on destroying mutated mtDNA as opposed to fixing mutations. Furthermore, TALEN technology cannot be utilised in individuals with a high mutation load as there is a risk that mtDNA copy numbers would reduce to detrimental levels in these individuals. A further advantage of DdCBEs is that the tool does not induce double-stranded DNA breaks. This is particularly useful for mtDNA genome editing since the mechanisms to repair such DNA damage are inefficient in mitochondria.
Precision genome editing for mtDNA with this new molecular tool shows great promise. Mok et al. have demonstrated that targeted base edits in mtDNA can be made in human cells with high specificity and low levels of off-target effects. Changes to respiration and oxidative phosphorylation rates were also validated in an mtDNA mutation associated disease model. The researchers clarify that this technology requires further research and optimisation, so therapeutical potential remains a future goal. However, for researchers of mitochondrial disease models, this new tool offers exciting potential and will enable fundamental questions in the field to be answered.
References: Mok, B., de Moraes, M., Zeng, J., Bosch, D., Kotrys, A., Raguram, A., Hsu, F., Radey, M., Peterson, S., Mootha, V., Mougous, J. and Liu, D., 2020. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 583(7817), pp.631-637.
Rees, H. and Liu, D., 2018. Base editing: precision chemistry on the genome and transcriptome of living cells. Nature Reviews Genetics, 19(12), pp.770-788.
Salter, J. and Smith, H., 2018. Modeling the Embrace of a Mutator: APOBEC Selection of Nucleic Acid Ligands. Trends in Biochemical Sciences, 43(8), pp.606-622.