Work

One‐step generation of heritable mitochondrial DNA multiplex‐engineered rats using DddA ‐derived cytosine base editor

Year: 2026

Type: article

Abstract: BACKGROUND: Mitochondrial DNA (mtDNA) mutations are implicated in a wide range of diseases, underscoring the need to elucidate the relationship between mtDNA mutations and disease pathology. These diseases are often characterized by the presence of multiple mutations; however, research has been hampered by the lack of suitable animal models carrying multiplex mtDNA mutations. Such models cannot be produced through traditional breeding due to matrilineal inheritance of mtDNA. METHODS: Based on the TALE-based mitochondrial genome editing tools, we generated rats harboring multiple mtDNA mutations by microinjecting mixed pairs of DdCBE plasmids into one-cell-stage zygotes. The efficiency of mtDNA editing and the potential off-target effects were assessed through deep sequencing and long reads sequencing methodologies. RESULTS: In this study, we established double- and triple-site mutant rats with the editing efficiencies of up to 58.5% and confirmed that these mutations can be stably transmitted through the germline. Moreover, our results demonstrated that DdCBE-mediated mtDNA multi-site editing of mtDNA exhibits minimal off-target effects in both the mitochondrial and nuclear genomes in vivo. CONCLUSION: This work represents the first successful generation of heritable multi-site mtDNA mutant rats, providing a valuable model for elucidating the pathophysiological mechanisms of mitochondrial disorders and for developing potential therapeutics.

Source: Animal Models and Experimental Medicine

DOI: 10.1002/ame2.70154