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

作者：Jiaxin Qiu, Haibo Wu, Qin Xie, Yuxiao Zhou, Yining Gao, Junbo Liu, Xueyi Jiang, Lun Suo, Yanping Kuang · 发表于：Frontiers in Bioengineering and Biotechnology · 年份：2024 · DOI：10.3389/fbioe.2024.1372211 · 被引用次数：9 · 研究领域：CRISPR and Genetic Engineering、Mitochondrial Function and Pathology、RNA and protein synthesis mechanisms

Introduction: 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. Methods: 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. Results: 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 8-11 (G1333) and 5’C 10-13 (G1397) for aC target, while 5’C 4-13 (G1333) and 5’C 5-14 (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. Discussion: In summary, this discovery helps us conceive the optimal strategy for accurate mtDNA editing, avoiding time- and effort-consumin...