Abstract
Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.
Highlights
The targeted manipulation of genes, involving either their knock-out, replacement or activation, is an attractive technology in the context of both basic and applied genetic research
We show that customized transcription activatorlike effector nucleases (TALENs) can induce targeted mutations in barley, and that these are faithfully transmitted to the progeny of primary mutant plants
Two single-copy lines (BPI 09 and PV 89) in which there was no segregation with respect to either the presence (Fig. S2a in File S1) or the expression (Fig. S2b in File S1) of gfp were used for the subsequent TALEN experiments
Summary
The targeted manipulation of genes, involving either their knock-out, replacement or activation, is an attractive technology in the context of both basic and applied genetic research. Engineered zinc-finger nucleases (ZFNs), transcription activatorlike effector nucleases (TALENs), homing endonucleases and RNA-guided nucleases (CRISPRs) have all been reported to be capable of inducing DSBs [1,2]; of these, the ZFNs have been the most frequently employed. TALENs are customizable fusion proteins between a specific DNA binding domain and FokI endonuclease, the former directing the nuclease to the userdefined genomic site. The binding domain is derived from transcription activator-like (TAL) effectors of pathogenic Xanthomonas spp., while the FokI nuclease domain introduces DSBs when present in a dimeric form, achieved by introducing a pair of TALEN units. As each repeat of the DNA binding domain of the TAL effector recognizes nucleotides in a largely context-independent manner [3,4], the selection of a target DNA motif and the subsequent customization of the TALEN is made relatively straightforward. Reliable platforms for the modular assembly of TALENs are provided by Golden Gate cloning [5,6,7,8], highthroughput solid phase assembly [9], and ligation-independent cloning techniques [10]
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