title: Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.
abstract: Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Nagaveni
authors.initials: N
authors.lastname: Budhagatapalli
authors.affiliation: Structural Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Twan
authors.initials: T
authors.lastname: Rutten
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Maia
authors.initials: M
authors.lastname: Gurushidze
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Stadt Seeland/OT Gatersleben, Germany hensel@ipk-gatersleben.de.
authors.forename: Goetz
authors.identifier: http://orcid.org/0000-0002-5539-3097
authors.initials: G
authors.lastname: Hensel
mesh_terms: D000483:Alleles; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004720:Endonucleases / Q000378:metabolism*; D005785:Gene Conversion; D018390:Gene Targeting / Q000379:methods*; D018745:Genome, Plant; D001467:Hordeum / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors; D004720:Endonucleases
keywords: allele conversion; barley; designer endonuclease; targeted gene modification