Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.
Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing.
复制标题
DOI：
10.1038/srep41478
复制
发表时间：
2017-02-02
影响因子：
4.6
通讯作者：
Nakagawa H
中科院分区：
文献类型：
--
作者：
Fukuda M;Umeno H;Nose K;Nishitarumizu A;Noguchi R;Nakagawa H
作为 DNA 诱变的替代方法，RNA 诱变有可能成为基础研究和应用生命科学的强大基因调控方法。腺苷到肌苷 (A-to-I) RNA 编辑会改变转录水平的遗传信息，是后生动物中普遍保守的重要生物过程。因此，利用细胞内RNA编辑机制可以实现多功能的RNA诱变方法。在此，我们报道了新型引导RNA，其能够通过引导编辑酶——人腺苷脱氨酶作用于RNA（ADAR）来诱导A至I突变。这些指导RNA成功地将A到I突变引入靶位点，这是由可重编程反义区域决定的。在 ADAR2 过度表达的细胞中，还可以通过简单地引入引导 RNA 来进行定点 RNA 编辑。我们的向导 RNA 框架为建立普遍适用的 RNA 诱变方法提供了基本见解。
As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.