Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice
Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice
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DOI：
10.1016/j.plaphy.2018.04.028
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发表时间：
2018-10-01
影响因子：
6.5
通讯作者：
Kondo, Akihiko
中科院分区：
文献类型：
--
作者：
Shimatani, Zenpei;Fujikura, Ushio;Kondo, Akihiko
CRISPR/Cas9系统是一种革命性的基因组编辑工具，可用于多种生物体的定向基因编辑。当与转录激活剂或 DNA 修饰酶等各种功能效应器结合时，Cas9 变体可用作分子归巢装置。 Target-AID 是核酸酶缺陷型 Cas9 与激活诱导的胞苷脱氨酶 (AID) 融合的合成复合物，可实现靶向核苷酸替换（C 至 T 或 G 至 A）。我们之前证明，通过 Target-AID 将所需的点突变引入目标基因可赋予水稻愈伤组织对除草剂的耐受性。为了充分开发 Target-AID 作为植物育种技术，引入突变的继承以及转基因的去除是必须解决的关键问题。在这里，我们报告了这种突变从愈伤组织向再生体及其后代的传递，从而产生了一代具有同时多重核苷酸取代的无选择标记（SMF）除草剂耐受性水稻植物。这些发现表明，Target-AID可以发展成为一种新型植物育种技术，结合复杂的靶向碱基编辑以及CRISPR/Cas9系统的简单性和多功能性，可以一次性改善多种性状。
The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus. Inheritance of the introduced mutations, as well as the removal of transgenes, are key issues that must be addressed in order to fully develop Target-AID as a plant breeding technique. Here we report the transmission of such mutations from the callus to regenerants and their progenies, leading to a generation of selectable marker-free (SMF) herbicide tolerant rice plants with simultaneous multiplex nucleotide substitutions. These findings demonstrate that Target-AID can be developed into novel plant breeding technology which enables improvement of multiplex traits at one time in combination with sophisticated targeted base editing with the simplicity and versatility of CRISPR/Cas9 system.