Pathogenic mitochondrial DNA (mtDNA) mutations often co‐exist with wild‐type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70–90%). Previously, our laboratory showed that mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY‐YIG homing nuclease from T4 phage (I‐TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev‐TALEs) could specifically bind and cleave mtDNA of patient‐derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged‐red fibers (MERRF). We tested two mitoTev‐TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev‐TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev‐TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.

致病性线粒体DNA（mtDNA）突变通常与野生型分子（mtDNA异质性）共存。当突变mtDNA的百分比很高时（70-90％），表型就会显现出来。以前，我们的实验室表明，针对线粒体的转录激活因子样效应核酸酶（mitoTALENs）可以消除异质细胞中的突变mtDNA。但是，mitoTALENs是二聚体并且相对较大，使得难以将其编码基因包装到病毒载体中，从而限制了其临床应用。来自T4噬菌体（I-TevI​​）的较小的单体GIY-YIG归巢核酸酶提供了潜在的替代方法。我们测试了分子杂交体（mitoTev‐TALE）是否能特异性结合并切割具有不同水平的m.8344A> G mtDNA点突变的患者源性杂交瘤的mtDNA，与带有参差不齐的红色纤维（MERRF）的肌阵挛性癫痫相关。我们测试了两种mitoTev-TALE设计，其中一种将mtDNA比例稳固地转化为野生型。当在具有高水平MERRF突变（91％突变）的克隆中测试此mitoTev-TALE时，异质性的改变导致氧化磷酸化功能的改善。mitoTev-TALE为mtDNA编辑提供了有效的体系结构，可以促进mtDNA编辑酶向受影响组织的治疗性输送。