Xie Qin, Wu Haibo, Long Hui, Xiao Caiwen, Qiu Jiaxin, Yu Weina, Jiang Xueyi, Liu Junbo, Zhang Shuo, Lyu Qifeng, Suo Lun, Kuang Yanping
Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
Mol Ther Nucleic Acids. 2024 Mar 11;35(2):102170. doi: 10.1016/j.omtn.2024.102170. eCollection 2024 Jun 11.
Efficient germline mtDNA editing is required to construct disease-related animal models and future gene therapy. Recently, the DddA-derived cytosine base editors (DdCBEs) have made mitochondrial genome (mtDNA) precise editing possible. However, there still exist challenges for editing some mtDNA sites in germline via zygote injection, probably due to the suspended mtDNA replication during preimplantation development. Here, we introduce a germline mtDNA base editing strategy: injecting DdCBEs into oocytes of secondary follicles, at which stage mtDNA replicates actively. With this method, we successfully observed efficient G-to-A conversion at a hard-to-edit site and also obtained live animal models. In addition, for those editable sites, this strategy can greatly improve the base editing efficiency up to 3-fold, which is more than that in zygotes. More important, editing in secondary follicles did not increase more the risk of off-target effects than that in zygotes. This strategy provides an option to efficiently manipulate mtDNA sites in germline, especially for hard-to-edit sites.
构建疾病相关动物模型和未来的基因治疗需要高效的生殖系线粒体DNA（mtDNA）编辑。最近，源自DddA的胞嘧啶碱基编辑器（DdCBEs）使线粒体基因组（mtDNA）的精确编辑成为可能。然而，通过受精卵注射在生殖系中编辑某些mtDNA位点仍然存在挑战，这可能是由于植入前发育过程中线粒体DNA复制暂停所致。在此，我们介绍一种生殖系mtDNA碱基编辑策略：将DdCBEs注射到次级卵泡的卵母细胞中，在此阶段mtDNA活跃复制。通过这种方法，我们成功地在一个难以编辑的位点观察到了高效的G到A转换，并且还获得了活体动物模型。此外，对于那些可编辑的位点，该策略可以将碱基编辑效率大大提高至3倍，高于受精卵中的编辑效率。更重要的是，在次级卵泡中进行编辑不会比在受精卵中增加更多的脱靶效应风险。该策略为有效操纵生殖系中的mtDNA位点提供了一种选择，特别是对于难以编辑的位点。
