Harnessing accurate mitochondrial DNA base editing mediated by DdCBEs in a predictable manner.

Mitochondrial diseases caused by mtDNA have no effective cures. Recently developed DddA-derived cytosine base editors (DdCBEs) have potential therapeutic implications in rescuing the mtDNA mutations. However, the performance of DdCBEs relies on designing different targets or improving combinations of split-DddA halves and orientations, lacking knowledge of predicting the results before its application. A series of DdCBE pairs for wide ranges of aC or tC targets was constructed, and transfected into Neuro-2a cells. The mutation rate of targets was compared to figure out the potential editing rules. It is found that DdCBEs mediated mtDNA editing is predictable: 1) aC targets have a concentrated editing window for mtDNA editing in comparison with tC targets, which at 5'C (G1333) and 5'C (G1397) for aC target, while 5'C (G1333) and 5'C (G1397) for tC target with 16bp spacer. 2) G1333 mediated C>T conversion at aC targets in DddA-half-specific manner, while G1333 and G1397 mediated C>T conversion are DddA-half-prefer separately for tC and aC targets. 3) The nucleotide adjacent to the 3' end of aC motif affects mtDNA editing. Finally, by the guidance of these rules, a cell model harboring a pathogenic mtDNA mutation was constructed with high efficiency and no bystander effects. In summary, this discovery helps us conceive the optimal strategy for accurate mtDNA editing, avoiding time- and effort-consuming optimized screening jobs.