Efficient Mitochondrial Genome Editing by CRISPR/Cas9.
Efficient Mitochondrial Genome Editing by CRISPR/Cas9.
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DOI：
10.1155/2015/305716
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发表时间：
2015
影响因子：
--
通讯作者：
Lee Y
中科院分区：
文献类型：
--
作者：
Jo A;Ham S;Lee GH;Lee YI;Kim S;Lee YS;Shin JH;Lee Y
CRISPR/Cas9系统已被广泛用于核DNA编辑，以产生突变或纠正特定的疾病等位基因。尽管CRISPR/Cas9的应用非常灵活，但目前还没有确定CRISPR/Cas9是否可以靶向线粒体进行mtDNA编辑。在这里，我们表明常规FLAG-Cas9可以定位于线粒体，以编辑线粒体DNA，其中sgRNA靶向线粒体基因组的特定位点。FLAG-Cas9与靶向Cox 1和Cox 3的gRNA一起表达导致特异性mtDNA基因座的切割。此外，我们观察到在mtDNA截短或CRISPR/Cas9切割后线粒体蛋白质稳态的破坏。为了克服FLAG-Cas9的非特异性分布，我们还创建了靶向大肠杆菌的Cas9（mitoCas 9）。这种新版本的Cas9仅定位于线粒体;与靶向mtDNA的gRNA的表达一起，存在mtDNA的特异性切割。MitoCas 9诱导的mtDNA及其转录的减少导致线粒体膜电位破坏和细胞生长抑制。这种mitoCas 9可以用于编辑mtDNA和gRNA表达载体，而不影响基因组DNA。在这项简短的研究中，我们证明了使用CRISPR/Cas9进行mtDNA编辑是可能的。此外，我们开发的特异性定位于线粒体的mitoCas 9应该有助于其用于线粒体基因组编辑。
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.