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Engineered Base Editing Tool Avoids Off-Target Mutations in Mitochondrial DNA

NEW YORK — Researchers have engineered a genome-editing tool to cut mitochondrial DNA more precisely, an improvement that could enable therapeutic editing.

While bacterial toxin DddA-derived cytosine base editors (DdCBEs) can be used to make targeted C-to-T conversions in nuclear and mitochondrial DNA, they have also been found to generate off-target mutations. DdCBEs are made up of different parts — a split interbacterial toxin DddAtox, a uracil glycosylase inhibitor, and custom transcriptome activator-like effector (TALE) DNA-binding proteins — and researchers from the Institute for Basic Science in South Korea suspected that those off-target effects might be driven by nonspecific TALE-DNA interactions or by spontaneous assembly of the split interbacterial toxin DddAtox.

As they reported Thursday in Nature Biotechnology, the researchers homed in on the split DddAtox halves and the interface where the dimers interact. By altering the residues there, they were able to generate base editors with reduced off-target effects, which they have dubbed HiFi-DdCBEs.

"Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications," senior author Jin-Soo Kim from the Institute for Basic Science and colleagues wrote.

The researchers first investigated the cause of the off-target mutations by the typical DdCBEs. They found that when halves of a split DddAtoxwere present without a TALE, they could still induce C-to-T conversions.

By examining the structure of DddAtox complex, the researchers identified amino acid residues on each half that interact with each other and then created a series of mutant DddAtox halves in which an interacting residue was replaced with a bulky alanine instead. That way, the two DddAtox halves cannot form a deaminase unit without a related TALE-DNA interaction.

One variant they generated, K1389A, was highly active when paired with a wild-type partner but was much more inefficient when paired with a TALE-free partner, showing a 38.5-fold difference in discrimination. Further, pairs containing the K1389A variant preferentially edited at the C8 position over the C9, C11, and C13 positions, while a wild-type pair would edit at any of those sites, suggesting this approach could limit off-target editing.

The researchers then tested the editing ability of these HiFi-DdCBEs variants in human cells and cataloged any on- or off-target base editing using mtDNA-wide whole-genome sequencing. Pairs containing the T1391A in 1397C, V1411A in 1397N, or T1413A in 1397N variants exhibited between 8.2- and 23.3-fold reduced off-target editing. Meanwhile, the on-target editing efficiency of the HiFi-DdCBEs were similar to wild-type DdCBE pairs with DddA6 or DddA11.

"Taken together, these results demonstrate that DdCBE off-target base editing in the mitochondrial genome can be largely avoided using our HiFi-DdCBEs with interface-engineered DddAtox split constructs," Kim and colleagues wrote.

Still, certain HiFi-DdCBEs might suit scientists' needs over others, depending on the situation. In particular, the researchers recommend using the T1391A variant in 1397N or 1333C if high specificity is needed more than high activity, and conversely the K1389A variant in 1397N or 1333C if high activity is preferred rather than high specificity.

They additionally cautioned that they investigated the off-target activity of HiFi-DdCBEs over the short term and that additional studies of their long-term activity will be needed.