MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK.
Nat Biomed Eng. 2023 May;7(5):692-703. doi: 10.1038/s41551-022-00968-1. Epub 2022 Dec 5.
The development of curative treatments for mitochondrial diseases, which are often caused by mutations in mitochondrial DNA (mtDNA) that impair energy metabolism and other aspects of cellular homoeostasis, is hindered by an incomplete understanding of the underlying biology and a scarcity of cellular and animal models. Here we report the design and application of a library of double-stranded-DNA deaminase-derived cytosine base editors optimized for the precise ablation of every mtDNA protein-coding gene in the mouse mitochondrial genome. We used the library, which we named MitoKO, to produce near-homoplasmic knockout cells in vitro and to generate a mouse knockout with high heteroplasmy levels and no off-target edits. MitoKO should facilitate systematic and comprehensive investigations of mtDNA-related pathways and their impact on organismal homoeostasis, and aid the generation of clinically meaningful in vivo models of mtDNA dysfunction.
治疗线粒体疾病的方法仍在发展中，此类疾病通常由线粒体 DNA（mtDNA）突变引起，会损害能量代谢和细胞内环境的其他方面。由于对其基础生物学的了解不够充分，以及细胞和动物模型的缺乏，此类疾病的治疗方法的发展受到了阻碍。在这里，我们报告了一种双链 DNA 脱氨酶衍生的胞嘧啶碱基编辑器文库的设计和应用，该文库经过优化，可精确靶向敲除小鼠线粒体基因组中的每个 mtDNA 蛋白编码基因。我们使用该文库（命名为 MitoKO）在体外产生近乎同质的敲除细胞，并生成具有高异质突变体水平且没有脱靶编辑的小鼠敲除体。MitoKO 应该有助于系统和全面地研究 mtDNA 相关途径及其对机体内环境稳定的影响，并有助于生成具有临床意义的 mtDNA 功能障碍的体内模型。
