Transfer of mitochondrial DNA into the nuclear genome during gene editing [mitoTALEN_ND4 PEM-seq]

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause broad transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial targeting editors, mitoTALEN and recently developed base editor DdCBE, can also induce widespread mtDNA integrations. However, we provide a practical solution to suppress mtDNA transfer by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings also shed light on the origins of mitochondrial-nuclear DNA segments. We employed primer-extension-mediated sequencing (PEM-seq) to study the level of mitochondrial DNA integration into the nuclear genome caused by genome editing.

线粒体作为细胞的能量工厂，其独特的DNA使其成为治疗遗传疾病的基因编辑潜在靶点。然而，基因组编辑对线粒体DNA（mtDNA）稳定性的影响仍不明晰。本研究揭示了此前未被认知的基因组编辑风险：无论是核基因组编辑还是线粒体基因组编辑，均可在多种细胞类型（包括人类细胞系、原代T细胞以及小鼠胚胎）中引发线粒体DNA片段向核基因组的广泛转移整合。此外，药物诱导的线粒体应激与mtDNA断裂会进一步加剧这一mtDNA向核基因组的转移整合过程。值得注意的是，本研究发现线粒体靶向编辑工具mitoTALEN以及新近开发的碱基编辑器DdCBE同样可诱导广泛的mtDNA整合事件。不过，本研究提出了一种切实可行的抑制mtDNA转移的方案：在DdCBE编辑过程中共表达TREX1或TREX2外切核酸酶。上述研究结果同时也为线粒体-核DNA片段的起源提供了新的科学阐释。本研究采用引物延伸介导测序（primer-extension-mediated sequencing, PEM-seq）技术，对基因组编辑所引发的线粒体DNA向核基因组整合的水平进行了系统性探究。