Secondary follicles enable efficient germline mitochondrial DNA base editing at hard-to-edit site

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 pre-implantation 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. Besides, for those editable sites, this strategy can also greatly improve the base editing efficiency up to threefold than that in zygotes. More importantly, editing in secondary follicles did not increase the risk of off-target effects than that in zygotes. Taken together, this strategy provides an option to efficiently manipulate mtDNA sites in germline, especially for hard-to-edit sites.