title: Stable gene replacement in barley by targeted double-strand break induction.
abstract: Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Stadt Seeland, Germany.
authors.forename: Koichi
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: Max-Planck-Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Breier
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Stadt Seeland, Germany.
authors.forename: Götz
authors.initials: G
authors.lastname: Hensel
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Stadt Seeland, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Stadt Seeland, Germany Faculty of Science and Central European Institute of Technology, Masaryk University, 61137 Brno, Czech Republic.
authors.forename: Ingo
authors.initials: I
authors.lastname: Schubert
authors.affiliation: Max-Planck-Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne, Germany reiss@mpipz.mpg.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Reiss
mesh_terms: D053903:DNA Breaks, Double-Stranded; D018390:Gene Targeting / Q000379:methods*; D017343:Genes, Plant; D056426:Genetic Loci; D001467:Hordeum / Q000235:genetics*; D040582:Inheritance Patterns / Q000235:genetics; D008957:Models, Genetic; D030821:Plants, Genetically Modified; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D014170:Transformation, Genetic
keywords: Barley; Hordeum vulgare; double-strand break induction; gene replacement; gene targeting; homology-directed DNA integration; precision genome engineering.