Engineering TALE-linked Deaminases to Facilitate Precision Adenine Base Editing in Mitochondrial DNA

DddA-derived cytosine base editors (DdCBEs) and transcription activator -like effector (TALE) -linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A -to -G -editing TALEDs but not C -to -T -editing DdCBEs induce tens of thousands of transcriptome-wide off -target edits in human cells. To avoid these unwanted RNA edits, 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 not only reduced RNA off -target edits by >99% but also minimized off -target mtDNA mutations and bystander edits at a target site. Unlike wildtype versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.