Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

Programmable deaminases, including bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs), zinc finger deaminases, and transcription activator-like effector (TALE)-linked deaminases (TALEDs), enable targeted C-to-T or A-to-G base editing of mitochondrial DNA (mtDNA) in cell lines and animals, which paves the way for modeling of mitochondrial genetic disorders and for developing novel therapeutic modalities. Here, we report that A-to-G base editing TALEDs but not C-to-T editing DdCBEs induce tens of thousands of transcriptome-wide off-target A-to-I edits in cultured human cells. To avoid these unwanted RNA off-target base conversions, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants reduced RNA off-target edits by > 99%, while retaining high DNA on-target editing activity. Furthermore, our TALED variants minimized off-target mutations in the mitochondrial genome as well as bystander edits at a target site. Importantly, unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we were able to obtain mtDNA-edited mice with m.T8585C and m.T8591C, pathogenic mutations associated with Leigh disease.