Generation of Gene-Edited Chrysanthemum morifolium Using Multicopy Transgenes as Targets and Markers
Generation of Gene-Edited Chrysanthemum morifolium Using Multicopy Transgenes as Targets and Markers
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DOI：
10.1093/pcp/pcw222
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发表时间：
2017-02-01
影响因子：
4.9
通讯作者：
Sasaki, Katsutomo
中科院分区：
文献类型：
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作者：
Kishi-Kaboshi, Mitsuko;Aida, Ryutaro;Sasaki, Katsutomo
最广泛使用的基因编辑技术-CRISPR/Cas9系统-采用了一种细菌单体DNA内切核酸酶，称为成簇规则间隔短回文重复序列（CRISPR）相关蛋白9（Cas9）和将Cas9引导至互补靶DNA的单向导RNA（sgRNA）。然而，将突变引入到更高的多倍体植物物种中，特别是对于没有基因组信息的物种，一直是困难的。杭白菊是重要的观赏植物之一，但它是一个六倍体，基因组庞大，而且缺乏全基因组信息。这些特征阻碍了菊花的基因组编辑。在本研究中，我们尝试使用CRISPR/Cas9系统进行基因编辑，将突变引入菊花。我们构建了表达来自辣椒的黄绿色荧光蛋白基因（CpYGFP）的转基因菊花植株，并以CpYGFP为靶点进行基因编辑。我们比较了花椰菜花叶病毒（CaMV）35 S启动子和欧芹泛素启动子在菊花愈伤组织中的活性，并选择欧芹泛素启动子来驱动Cas9。我们选择了两种sgRNA靶向CpYGFP基因中的不同位置，并获得了含有突变的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，含有突变的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序列中含有突变的CRISPR-CpYGFP-菊花芽。这是在菊花中使用CRISPR/Cas9系统进行基因编辑的第一份报告，为菊花基因组编辑提供了线索。
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.