SUNGKYUNKWAN UNIVERSITY SCHOOL OF MEDICINE

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deam- inases (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 transcrip- tome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the sub- strate-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 wild- type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse em- bryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.