新加坡国立大学Jin-Soo Kim等研究人员合作发现，工程化TALE连接的脱氨酶可促进线粒体DNA中腺嘌呤碱基的精确编辑。相关论文于2024年1月4日发表在《细胞》杂志上。
研究人员表示，DddA衍生的胞嘧啶碱基编辑器（DdCBE）和转录激活剂样效应物（TALE）连接的脱氨酶（TALED）可催化真核细胞中线粒体DNA（mtDNA）的靶向碱基编辑，这种方法有助于线粒体遗传疾病的建模和新型治疗方法的开发。
研究人员报告了在人体细胞中，A-to-G编辑的TALED而非C-to-T编辑的DdCBE会诱导数以万计的转录组范围的脱靶编辑。为了避免这些不必要的RNA编辑，研究人员设计了TALED中脱氧腺嘌呤脱氨酶TadA8e的底物结合位点，并创造了具有微调脱氨酶活性的TALED变体。研究人员设计的TALED变体不仅能将RNA脱靶编辑减少99%以上，还能最大限度地减少靶点上的脱靶mtDNA突变和旁观者编辑。
与野生型不同，这种TALED变体没有细胞毒性，也不会导致小鼠胚胎发育停滞。因此，研究人员获得了与利氏综合征有关的致病性mtDNA突变的小鼠，这些小鼠的心率降低。
附：英文原文
Title: Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA
Author: Sung-Ik Cho, Kayeong Lim, Seongho Hong, Jaesuk Lee, Annie Kim, Chae Jin Lim, Seungmin Ryou, Ji Min Lee, Young Geun Mok, Eugene Chung, Sanghun Kim, Seunghun Han, Sang-Mi Cho, Jieun Kim, Eun-Kyoung Kim, Ki-Hoan Nam, Yeji Oh, Minkyung Choi, Tae Hyeon An, Kyoung-Jin Oh, Seonghyun Lee, Hyunji Lee, Jin-Soo Kim
Issue&Volume: 2024/01/04
Abstract: 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.
DOI: 10.1016/j.cell.2023.11.035
Source: https://www.cell.com/cell/fulltext/S0092-8674(23)01321-1