title: Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.
abstract: Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.
authors.affiliation: Plant Genome Engineering Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Nishizawa-Yokoi
authors.forename: Masaki
authors.initials: M
authors.lastname: Endo
authors.forename: Namie
authors.initials: N
authors.lastname: Ohtsuki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Saika
authors.forename: Seiichi
authors.initials: S
authors.lastname: Toki
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant; D053209:Herbicide Resistance / Q000235:genetics*; D006540:Herbicides / Q000494:pharmacology*; D016296:Mutagenesis; D012275:Oryza / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics; D017354:Point Mutation; D019895:Transposases / Q000235:genetics
chemical_list: D006540:Herbicides; D000095:Acetolactate Synthase; D019895:Transposases
keywords: Oryza sativa; acetolactate synthase; cleistogamy 1; gene targeting; marker excision; piggyBac transposon; technical advance