title: Generation of Gene-Edited Chrysanthemum morifolium Using Multicopy Transgenes as Targets and Markers.
abstract: 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.
authors.affiliation: Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Kishi-Kaboshi
authors.affiliation: National Institute of Floricultural Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Aida
authors.affiliation: Japan International Research Center for Agricultural Sciences (JIRCAS), Ohwashi, Tsukuba, Ibaraki, Japan.|Graduate School of Agricultural and Life Sciences, Institute of Sustainable Agro-ecosystem Services (ISAS), The University of Tokyo, Midoricho, Nishitokyo, Tokyo, Japan.
authors.forename: Katsutomo
authors.initials: K
authors.lastname: Sasaki
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D017796:Caulimovirus / Q000235:genetics; D031202:Chrysanthemum / Q000235:genetics* / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D017393:RNA Editing / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR/Cas9 system; Chrysanthemum morifolium; CpYGFP; Gene editing