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

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.

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.

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.

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.