Hussain Syed-Rehan A, Yalvac Mehmet E, Khoo Benedict, Eckardt Sigrid, McLaughlin K John
Center for Molecular and Human Genetics, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH, United States.
Center for Clinical and Translational Research, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH, United States.
Front Genet. 2021 Apr 6;12:627050. doi: 10.3389/fgene.2021.627050. eCollection 2021.
Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.
使用CRISPR-Cas9系统对线粒体基因组进行基因编辑极具挑战性，主要原因是向导RNA和Cas9酶复合物向线粒体的递送效率低下。在本研究中，我们通过将靶向烟酰胺腺嘌呤二核苷酸-泛醌氧化还原酶链4（ND4）的向导RNA附加到RNA转运衍生的茎环元件（RP环）上，并表达带有先前线粒体定位序列的Cas9酶，从而能够在线粒体DNA中进行基因编辑。我们观察到RP环向导RNA在线粒体中的共定位，并且在其ND4序列中携带11205G变体的细胞中，ND4表达显著降低，同时线粒体DNA水平也随之下降。这项概念验证研究表明，添加了茎环元件的单向导RNA可以转运到线粒体中，并与Cas9发生功能相互作用，从而介导序列特异性线粒体DNA切割。利用这种针对线粒体DNA的新方法，我们的结果进一步证明，CRISPR-Cas9介导的基因编辑可能有潜力用于治疗线粒体相关疾病。
