title: Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut.
abstract: Genome modification by homology-directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR-mediated gene exchange of expression cassettes in tobacco BY-2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7-kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4-kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR-mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin-resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN-based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Schneider
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Andreas
authors.identifier: http://orcid.org/0000-0002-2057-4111
authors.initials: A
authors.lastname: Schiermeyer
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Dolls
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Koch
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Herwartz
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Kirchhoff
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Russell
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: Zehui
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Corbin
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Sastry-Dent
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: W Michael
authors.initials: WM
authors.lastname: Ainley
authors.affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Webb
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Helga
authors.initials: H
authors.lastname: Schinkel
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D015139:Blotting, Southern; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D018390:Gene Targeting / Q000379:methods*; D007613:Kanamycin Resistance / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D059828:Plant Cells; D030821:Plants, Genetically Modified; D059767:Recombinational DNA Repair / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D016335:Zinc Fingers; D000097573:Red Fluorescent Protein
chemical_list: D003851:Deoxyribonucleases; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: direct DNA delivery; flow cytometry; gene targeting; homologous recombination; tobacco BY-2 cell suspension cultures