Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy
Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy
复制标题
DOI：
10.1038/gt.2017.90
复制
发表时间：
2017-12-01
期刊：
影响因子：
5.1
通讯作者：
Tsukahara, T.
中科院分区：
文献类型：
--
作者：
Azad, Md T. A.;Bhakta, S.;Tsukahara, T.
定点RNA编辑是纠正基因序列并最终调节蛋白质功能的重要技术。在这项研究中，我们设计了腺苷脱氨酶作用于RNA的脱氨酶结构域（ADAR 1）和MS 2系统，以靶向特异性腺苷，目的是在RNA水平上纠正G到A突变。为此，ADAR 1脱氨酶结构域融合在RNA结合蛋白MS 2的下游，该蛋白对MS 2 RNA具有亲和力。为了将编辑引导至特定靶标，我们设计了与靶RNA互补的引导RNA。向导RNA将ADAR 1脱氨酶引导到所需的编辑位点，在那里它将腺苷转化为肌苷。为了提供原理证明，我们使用了增强型绿色荧光蛋白（EGFP）的等位基因，该等位基因在第58个氨基酸（TGG）编码Trp，突变为琥珀色（TAG）或赭色（TAA）终止密码子。在HEK-293细胞中，我们的系统可以将终止密码子转换为通读密码子，从而打开荧光。我们通过限制性片段长度多态性分析和测序证实了DNA水平编辑的特异性，并通过蛋白质印迹证实了蛋白质水平编辑的特异性。该酶系的编辑效率接近5%。我们相信这个系统可以用来治疗由G到A点突变引起的遗传疾病。
Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to targetspecific adenosines, with the goal of correcting G-to-A mutations at the RNA level. For this purpose, the ADAR1 deaminase domain was fused downstream of the RNA-binding protein MS2, which has affinity for the MS2 RNA. To direct editing to specific targets, we designed guide RNAs complementary to target RNAs. The guide RNAs directed the ADAR1 deaminase to the desired editing site, where it converted adenosine to inosine. To provide proof of principle, we used an allele of enhanced green fluorescent protein (EGFP) bearing a mutation at the 58th amino acid (TGG), encoding Trp, into an amber (TAG) or ochre (TAA) stop codon. In HEK-293 cells, our system could convert stop codons to read-through codons, thereby turning on fluorescence. We confirmed the specificity of editing at the DNA level by restriction fragment length polymorphism analysis and sequencing, and at the protein level by western blotting. The editing efficiency of this enzyme system was similar to 5%. We believe that this system could be used to treat genetic diseases resulting from G-to-A point mutations.