title: Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases.
abstract: We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.
authors.affiliation: Department of Agronomy and Plant Genetics, University of Minnesota, St Paul, Minnesota 55108, USA.
authors.forename: Shaun J
authors.initials: SJ
authors.lastname: Curtin
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
authors.forename: William J
authors.initials: WJ
authors.lastname: Haun
authors.forename: Colby
authors.initials: C
authors.lastname: Starker
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Baltes
authors.forename: Deepak
authors.initials: D
authors.lastname: Reyon
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Dahlborg
authors.forename: Mathew J
authors.initials: MJ
authors.lastname: Goodwin
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Coffman
authors.forename: Drena
authors.initials: D
authors.lastname: Dobbs
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stupar
mesh_terms: D001483:Base Sequence; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism*; D020131:Genes, Duplicate / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics*; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D040582:Inheritance Patterns / Q000235:genetics; D020407:Internet; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D018517:Plant Roots / Q000235:genetics; D016133:Polymerase Chain Reaction; D013025:Glycine max / Q000235:genetics*; D019076:Transgenes / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D004720:Endonucleases