DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome

• Published in: Molecular therapy. Nucleic acids Vol. 27; pp. 73 - 80
• Main Authors: Guo, Jiayin, Chen, Xiaoxu, Liu, Zhiwei, Sun, Haifeng, Zhou, Yu, Dai, Yichen, Ma, Yu'e, He, Lei, Qian, Xuezhen, Wang, Jianying, Zhang, Jie, Zhu, Yichen, Zhang, Jun, Shen, Bin, Zhou, Fei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.03.2022; American Society of Gene & Cell Therapy; Elsevier
• Subjects: base editing; DdCBE; mitochondrial disorder; mouse model; mtDNA; Original; DdCBE; mouse model; mtDNA; base editing; mitochondrial disorder
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2021.11.016

Critical mutations of mitochondrial DNA (mtDNA) generally lead to maternally inheritable diseases that affect multiple organs and systems; however, it was difficult to alter mtDNA in mammalian cells to intervene in or cure mitochondrial disorders. Recently, the discovery of DddA-derived cytosine base editor (DdCBE) enabled the precise manipulation of mtDNA. To test its feasibility for in vivo use, we selected several sites in mouse mtDNA as DdCBE targets to resemble the human pathogenic mtDNA G-to-A mutations. The efficiency of DdCBE-mediated mtDNA editing was first screened in mouse Neuro-2A cells and DdCBE pairs with the best performance were chosen for in vivo targeting. Microinjection of the mRNAs of DdCBE halves in the mouse zygotes or 2-cell embryo successfully generated edited founder mice with a base conversion rate ranging from 2.48% to 28.51%. When backcrossed with wild-type male mice, female founders were able to transmit the mutations to their offspring with different mutation loads. Off-target analyses demonstrated a high fidelity for DdCBE-mediated base editing in mouse mtDNA both in vitro and in vivo. Our study demonstrated that the DdCBE is feasible for generation of mtDNA mutation models to facilitate disease study and for potential treatment of mitochondrial disorders. [Display omitted] By intracytoplasmic mRNA injection into mouse zygotes or 2-cell embryos, DddA-derived cytosine base editor (DdCBE) achieves efficient and precise installation of human pathogenic mtDNA variants with relatively low off-target editing in founders. The mtDNA mutations can be transmitted maternally, producing offspring with different mutation loads.