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Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double strand breaks in barley

Nagaveni Budhagatapalli, Twan Rutten, Maia Gurushidze, Jochen Kumlehn, Goetz Hensel
doi: https://doi.org/10.1101/019893
Nagaveni Budhagatapalli
aPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland/OT Gatersleben, Germany
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Twan Rutten
bStructural Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland/OT Gatersleben, Germany
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Maia Gurushidze
aPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland/OT Gatersleben, Germany
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Jochen Kumlehn
aPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland/OT Gatersleben, Germany
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Goetz Hensel
aPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Stadt Seeland/OT Gatersleben, Germany
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  • For correspondence: hensel@ipk-gatersleben.de
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ABSTRACT

Transcription activator-like effector nucleases (TALENs) 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 in order to induce double strand breaks (DSBs) and thereby modify the host genome on a predefined target site via non-homologous 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 TALENs 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 YFP accumulation in about 3 out of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. Predictable genetic modifications comprising only a few genomic base pairs rather than entire genes are of particular practical relevance, because they might not fall under the European regulation of genetically engineered organisms. 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.

  • Abbreviations

    HDR
    Homology-directed repair;
    NHEJ
    Non-homologous end joining;
    RGEN
    RNA guided genome editing nucleases;
    GT
    Gene targeting;
    DSBs
    Double strand breaks.
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted May 28, 2015.
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    Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double strand breaks in barley
    Nagaveni Budhagatapalli, Twan Rutten, Maia Gurushidze, Jochen Kumlehn, Goetz Hensel
    bioRxiv 019893; doi: https://doi.org/10.1101/019893
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    Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double strand breaks in barley
    Nagaveni Budhagatapalli, Twan Rutten, Maia Gurushidze, Jochen Kumlehn, Goetz Hensel
    bioRxiv 019893; doi: https://doi.org/10.1101/019893

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