Publication details

Stable gene replacement in barley by targeted double-strand break induction

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Year of publication 2016
Type Article in Periodical
Magazine / Source Journal of Experimental Botany
MU Faculty or unit

Central European Institute of Technology

Field Genetics and molecular biology
Keywords Barley; double-strand break induction; gene replacement; gene targeting; homology-directed DNA integration; Hordeum vulgare; precision genome engineering
Description Using double-strand break induction in the crop plant barley, standard transformation technology allows gene replacement to occur at frequencies suitable for routine application.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.
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