In planta gene targeting

Abstract

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a sitespecific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.plant biotechnology | plant breeding | gene technology | double-strand-break repair S ince the first report on gene targeting (GT) in plants was published (1), various approaches were tested to improve the efficiency of the method (2-5), which has been summarized in recent reviews (6, 7). We were able to demonstrate that the integration of a transfer DNA (T-DNA) by homologous recombination (HR) into a specific locus could be enhanced by two orders of magnitude via double-strand-break (DSB) induction using a site-specific endonuclease (8). More recently, by the use of zinc finger nucleases (9), which, in principle, can be used to induce a DSB at any genomic site, endogenous loci have been targeted in Arabidopsis (10), tobacco (11), and maize (12) at high frequencies (13). Some time ago, a method for in vivo targeting was developed in Drosophila. A stably integrated donor precursor molecule is first circularized by the FLP recombinase and subsequently linearized via cutting a single I-SceI recognition site, generating the actual GT vector (14). However, such a technique has not been successfully transferred to plants. We have previously shown that DNA can be efficiently excised from the genome in planta by the use of a site-specific endonuclease (15). To test whether the combination of this approach with DSB-induced recombination might lead to an efficient GT system, that is independent of transformation, we performed a proof-of-concept (POC) experiment in Arabidopsis using I-SceI (16) as a sitespecific nuclease. ResultsGenerating Homozygous Single-Copy GT Lines. Our in planta GT system is based on three different constructs (two shown in Fig. 1A) that were transformed independently by floral dipping. The target locus contains a truncated β-glucuronidase (GUS) gene (uidA) that can be restored via GT. DSB induction at the two ISceI recognition sites flanking a kanamycin-resistance gene would result in excision of the kanamycin-resistance gene and in activation of the target locus for HR. Th...

Gene targeting in plants: 25 years later

“…For many crop plants, it is still difficult to obtain and regenerate to fertility even a single transgenic line. Therefore we recently developed a GT technique that should be applicable to all transformable plant species, even if the transformation efficiency is extremely low (Fauser et al, 2012). The basic idea behind the strategy is that, if one were able to perform the targeting reaction during plant development, then the progeny should carry targeted modifications.…”

In Planta Somatic Homologous Recombination Assay Revisited: A Successful and Versatile, but Delicate Tool

“…Experimental introduction of site-specific DSBs in target DNA by the use of the endonucleases HO or I-SceI increased the efficiency of SHR by up to two orders of magnitude (Chiurazzi et al, 1996;Orel et al, 2003). Enzymatic DSB induction also led to tremendous improvements in gene targeting (Puchta et al, 1996;Fauser et al, 2012). The fact that introduction of DSBs in the recombination partner(s) strongly increased the frequency of HR, including gene targeting, clearly speaks in favor of a mechanism that can be explained solely by HR.…”

Genome Editing Using Crispr/Cas System: New Era Genetic Technology in Agriculture to Boost Crop Output

“…On the in spite of these outcomes, a researchers utilizing Cas9 in rice found a putative off-target site to be changed in 1.6 % of the aggregate plants, despite the fact that this was as yet five times less continuous than the on-target site. For decreasing those offtarget transformations most recent enhancements to the CRISPR framework have been made using truncated gRNA (truncated inside the crRNA-inferred grouping) or by including two additional guanine (G) nucleotides to the 5' end [12,13]. Analysts have likewise endeavored to limit off-target impacts in another route by using "matched nickases".…”