Cargando…

Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley

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 br...

Descripción completa

Detalles Bibliográficos
Autores principales: Budhagatapalli, Nagaveni, Rutten, Twan, Gurushidze, Maia, Kumlehn, Jochen, Hensel, Goetz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555222/
https://www.ncbi.nlm.nih.gov/pubmed/26153077
http://dx.doi.org/10.1534/g3.115.018762
_version_ 1782388157982965760
author Budhagatapalli, Nagaveni
Rutten, Twan
Gurushidze, Maia
Kumlehn, Jochen
Hensel, Goetz
author_facet Budhagatapalli, Nagaveni
Rutten, Twan
Gurushidze, Maia
Kumlehn, Jochen
Hensel, Goetz
author_sort Budhagatapalli, Nagaveni
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4555222
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Genetics Society of America
record_format MEDLINE/PubMed
spelling pubmed-45552222015-09-01 Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley Budhagatapalli, Nagaveni Rutten, Twan Gurushidze, Maia Kumlehn, Jochen Hensel, Goetz G3 (Bethesda) Investigations 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. Genetics Society of America 2015-07-06 /pmc/articles/PMC4555222/ /pubmed/26153077 http://dx.doi.org/10.1534/g3.115.018762 Text en Copyright © 2015 Budhagatapalli et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Budhagatapalli, Nagaveni
Rutten, Twan
Gurushidze, Maia
Kumlehn, Jochen
Hensel, Goetz
Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title_full Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title_fullStr Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title_full_unstemmed Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title_short Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley
title_sort targeted modification of gene function exploiting homology-directed repair of talen-mediated double-strand breaks in barley
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555222/
https://www.ncbi.nlm.nih.gov/pubmed/26153077
http://dx.doi.org/10.1534/g3.115.018762
work_keys_str_mv AT budhagatapallinagaveni targetedmodificationofgenefunctionexploitinghomologydirectedrepairoftalenmediateddoublestrandbreaksinbarley
AT ruttentwan targetedmodificationofgenefunctionexploitinghomologydirectedrepairoftalenmediateddoublestrandbreaksinbarley
AT gurushidzemaia targetedmodificationofgenefunctionexploitinghomologydirectedrepairoftalenmediateddoublestrandbreaksinbarley
AT kumlehnjochen targetedmodificationofgenefunctionexploitinghomologydirectedrepairoftalenmediateddoublestrandbreaksinbarley
AT henselgoetz targetedmodificationofgenefunctionexploitinghomologydirectedrepairoftalenmediateddoublestrandbreaksinbarley