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.

Leibniz Institute of Plant Genetics and Crop Plant Research D 06466 Gatersleben Stadt Seeland Germany

Leibniz Institute of Plant Genetics and Crop Plant Research D 06466 Gatersleben Stadt Seeland Germany Faculty of Science and Central European Institute of Technology Masaryk University 61137 Brno Czech Republic

Max Planck Institute for Plant Breeding Research Carl von Linne Weg 10 D 50829 Cologne Germany

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Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches