Kishi-Kaboshi Mitsuko, Aida Ryutaro, Sasaki Katsutomo
Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
National Institute of Floricultural Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Plant Cell Physiol. 2017 Feb 1;58(2):216-226. doi: 10.1093/pcp/pcw222.
The most widely used gene editing technology-the CRISPR/Cas9 system-employs a bacterial monomeric DNA endonuclease known as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) and single-guide RNA (sgRNA) that directs Cas9 to a complementary target DNA. However, introducing mutations into higher polyploid plant species, especially for species without genome information, has been difficult. Chrysanthemum morifolium (chrysanthemum) is one of the most important ornamental plants, but it is a hexaploid with a large genome; moreover, it lacks whole-genome information. These characteristics hinder genome editing in chrysanthemum. In the present study, we attempted to perform gene editing using the CRISPR/Cas9 system to introduce mutations into chrysanthemum. We constructed transgenic chrysanthemum plants expressing the yellowish-green fluorescent protein gene from Chiridius poppei (CpYGFP) and targeted CpYGFP for gene editing. We compared the activity of a Cauliflower mosaic virus (CaMV) 35S promoter and parsley ubiquitin promoter in chrysanthemum calli and chose the parsley ubiquitin promoter to drive Cas9. We selected two sgRNAs to target different positions in the CpYGFP gene and obtained transgenic calli containing mutated CpYGFP genes (CRISPR-CpYGFP-chrysanthemum). A DNA sequencing analysis and fluorescence observations indicated that cells containing the mutated CpYGFP gene grew independently of cells containing the original CpYGFP gene in one callus. We finally obtained the CRISPR-CpYGFP-chrysanthemum shoot containing a mutation in the CpYGFP sequence. This is the first report of gene editing using the CRISPR/Cas9 system in chrysanthemum and sheds light on chrysanthemum genome editing.
应用最为广泛的基因编辑技术——CRISPR/Cas9系统，采用一种细菌单体DNA核酸内切酶，即成簇规律间隔短回文重复序列（CRISPR）相关蛋白9（Cas9）和引导Cas9靶向互补目标DNA的单向导RNA（sgRNA）。然而，在多倍体植物中引入突变一直颇具难度，尤其是对于那些没有基因组信息的物种而言。菊花是最重要的观赏植物之一，却是具有庞大基因组的六倍体；此外，它还缺乏全基因组信息。这些特性阻碍了对菊花进行基因组编辑。在本研究中，我们尝试利用CRISPR/Cas9系统对菊花进行基因编辑以引入突变。我们构建了表达来自波氏棘隙吸虫的黄绿色荧光蛋白基因（CpYGFP）的转基因菊花植株，并针对CpYGFP进行基因编辑。我们比较了花椰菜花叶病毒（CaMV）35S启动子和欧芹泛素启动子在菊花愈伤组织中的活性，选择了欧芹泛素启动子来驱动Cas9。我们选择了两个sgRNA靶向CpYGFP基因的不同位置，获得了含有突变CpYGFP基因的转基因愈伤组织（CRISPR-CpYGFP-菊花）。DNA测序分析和荧光观察表明，在一个愈伤组织中，含有突变CpYGFP基因的细胞独立于含有原始CpYGFP基因的细胞生长。我们最终获得了在CpYGFP序列中存在突变的CRISPR-CpYGFP-菊花苗。这是关于在菊花中使用CRISPR/Cas9系统进行基因编辑的首次报道，为菊花基因组编辑提供了思路。
