title: Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.
abstract: Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.
authors.affiliation: NARO Institute of Fruit Tree and Tea Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan.
authors.forename: Chikako
authors.initials: C
authors.lastname: Nishitani
authors.affiliation: NARO Institute of Fruit Tree and Tea Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan.
authors.forename: Narumi
authors.initials: N
authors.lastname: Hirai
authors.affiliation: Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
authors.forename: Sadao
authors.initials: S
authors.lastname: Komori
authors.affiliation: NARO Institute of Fruit Tree and Tea Science, 92-24, Nabeyashiki, Shimokuriyagawa, Morioka, Iwate 020-0123, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Wada
authors.affiliation: NARO Institute of Fruit Tree and Tea Science, 92-24, Nabeyashiki, Shimokuriyagawa, Morioka, Iwate 020-0123, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Okada
authors.affiliation: Faculty of Bioscience and Bioindustry, Tokushima University, 2-1 Josanjima, Tokushima 770-8513, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Osakabe
authors.affiliation: NARO Institute of Fruit Tree and Tea Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Faculty of Bioscience and Bioindustry, Tokushima University, 2-1 Josanjima, Tokushima 770-8513, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Osakabe
mesh_terms: D064113:CRISPR-Cas Systems; D002678:Chimera; D000072669:Gene Editing / Q000379:methods*; D018745:Genome, Plant; D027845:Malus / Q000235:genetics*; D000069600:Plant Breeding / Q000379:methods*