title: Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.
abstract: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.
authors.affiliation: Institute of Plant Sciences, Agricultural Research Organization, P.O. Box 6, 50250, Bet-Dagan, Israel.
authors.forename: Reut
authors.initials: R
authors.lastname: Peer
authors.forename: Gil
authors.initials: G
authors.lastname: Rivlin
authors.forename: Sara
authors.initials: S
authors.lastname: Golobovitch
authors.forename: Moshe
authors.initials: M
authors.lastname: Lapidot
authors.forename: Amit
authors.initials: A
authors.lastname: Gal-On
authors.forename: Alexander
authors.initials: A
authors.lastname: Vainstein
authors.forename: Tzvi
authors.initials: T
authors.lastname: Tzfira
authors.forename: Moshe A
authors.initials: MA
authors.lastname: Flaishman
mesh_terms: D004720:Endonucleases / Q000235:genetics*; D030681:Ficus / Q000201:enzymology / Q000235:genetics*; D005638:Fruit / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D018745:Genome, Plant / Q000235:genetics*; D023281:Genomics; D027845:Malus / Q000201:enzymology / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D016415:Sequence Alignment; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D010940:Plant Proteins; D004720:Endonucleases