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.
Highlights
We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs)
The ZFN is integrated into the soybean chromosome along with genes from A. rhizogenes that promote root development
Transgenic hairy roots can be obtained within two weeks of transformation and we reasoned they would be useful for testing the efficacy of ZFN mutagenesis prior to whole-plant transformation and provide an effective means of rapidly screening ZFN function at endogenous targets
Summary
We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). We 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. Upon successful dimerization of the FokI monomers, a double-stranded break is generated in the spacer sequence between both ZFA binding sites. This double-stranded break subsequently stimulates the cellular DNA repair pathways, which include the error-prone nonhomologous endjoining and the homology-directed repair. The nonhomologous end-joining pathway ligates double-stranded breaks in DNA, often introducing small nucleotide insertions and deletions (indels) at the target DNA site that can disrupt the gene’s reading frame
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