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

• Published in: Cell 2024-01, Vol.187 (1), p.95-109.e26
• Main Authors: Cho, Sung-Ik, Lim, Kayeong, Hong, Seongho, Lee, Jaesuk, Kim, Annie, Lim, Chae Jin, Ryou, Seungmin, Lee, Ji Min, Mok, Young Geun, Chung, Eugene, Kim, Sanghun, Han, Seunghun, Cho, Sang-Mi, Kim, Jieun, Kim, Eun-Kyoung, Nam, Ki-Hoan, Oh, Yeji, Choi, Minkyung, An, Tae Hyeon, Oh, Kyoung-Jin, Lee, Seonghyun, Lee, Hyunji, Kim, Jin-Soo
• Format: Article
• Language: English
• Subjects: Adenine; Animals; Cytosine; DNA, Mitochondrial - genetics; Gene Editing; Humans; Mice; RNA; Transcription Activator-Like Effectors
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2023.11.035

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 wild-type 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.