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

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.