Zakirova E G, Vyatkin Y V, Verechshagina N A, Muzyka V V, Mazunin I O, Orishchenko K E
Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
AcademGene Ltd., Novosibirsk, Russia.
Vavilovskii Zhurnal Genet Selektsii. 2020 Aug;24(5):512-518. doi: 10.18699/VJ20.643.
It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.
长期以来，人们一直知道线粒体基因组结构缺陷会导致各种神经肌肉和神经退行性疾病。然而，目前尚无治疗线粒体疾病的有效方法。治疗此类疾病的主要问题与线粒体DNA（mtDNA）异质性有关。这意味着由于线粒体基因组的高拷贝数，mtDNA的突变拷贝与野生型分子在同一细胞器中共存。线粒体疾病的临床症状及其表现程度直接取决于细胞中突变mtDNA分子的数量。减少突变不良影响的可能方法是将异质性水平转向野生型mtDNA分子。基于这一想法，已经为此开发了几种基于TALE和ZF核酸酶的基因治疗方法。然而，此类系统蛋白质结构域的构建是一个相当漫长且费力的过程。同时，CRISPR/Cas9系统与蛋白质系统有根本不同，因为它易于使用、效率高且作用机制不同。CRISPR/Cas9系统的所有特性和能力使其成为线粒体基因工程中有前景的工具。在本文中，我们首次证明通过在gRNA支架中整合特定的线粒体导入决定因素来修饰gRNA，在体外不会影响gRNA/Cas9复合物的活性。
