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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 Extensive analysis will identify the nature of the observed recombination events in order to discriminate between ‘true’ and ectopic gene targeting events. We recently extended the strategy to different promoters allowing activation of our gene targeting system in other specific cell types where homologous recombination is suspected to be the predominant recombination mechanism. ---------------------------------------------------------------------------------------------------------------------------------- T - 41. Meganucleases with tailored cleavage specificity can induce efficient homologous gene targeting Sylvain Arnould, Jean-Pierre Cabaniols, Philippe Duchateau, Aymeric Duclert, Jean-Charles Epinat, Agnès Gouble, Sylvestre Grizot, Christophe Perez, Julianne Smith, Frédéric Pâques Scientific Department, Cellectis S.A., 93 235 Romainville, France Homologous gene targeting is the best way to modify a genome in a precise and rational way, but has proved to be very inefficient in plants. This limit can be alleviated by meganucleases, sequence-specific endonucleases recognizing large (>12 bp) cleavage sites. These proteins can stimulate homologous gene targeting by a 1000-fold factor or induce mutagenesis in the vicinity of their target site, and these findings have opened novel perspectives for genome engineering in plants. However, the use of this technology has long been limited by the repertoire of natural meganucleases: the probability of finding a sequence cleaved by a natural meganuclease in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. Given their exceptional specificity, natural meganucleases should provide ideal scaffolds to derive genome engineering tools.We have developed a combinatorial approach to redesign the DNA-binding interface of I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of meganucleases. First, we collect large numbers of locally engineered I-CreI derivatives with altered specificity. Second, we assemble these mutants into entirely redesigned endonucleases binding a priori chosen targets. The engineered proteins keep the essential properties of natural meganucleases in terms of folding, activity and specificity, and can be used to induce recombination and site directed mutagenesis in chromosomal sequences. Thus, our strategy provides the means to engineer a very large number of sequences in a very specific way. We will illustrate both the protein engineering and genome engineering steps. ---------------------------------------------------------------------------------------------------------------------------------- T - 42. Toward zinc finger nucleases-mediated gene targeting in plants Andriy Tovkach, Vardit Zeevi, Tzvi Tzfira Department of Molecular, Cellular, & Developmental Biology, University of Michigan, 48109-1048 Ann Arbor, MI, United States Double-strand breaks (DSBs) in plant genomes are typically repaired by the plant nonhomologous end-joining (NHEJ) machinery, which usually leads to local mutagenesis due to small deletions at the repair site. We have shown that double stranded T-DNA molecules preferentially integrate into DSBs in transgenic plants carrying an I-SceI endonuclease recognition site which, upon cleavage with I-SceI, generates a DSB. In addition, using AscI, an 8 base cutter recognizes ca. 80 sites in the Arabidopsis genome we discovered that DSBs acts as attractants of T-DNA molecules at different genomic locations. This phenomenon could potentially be used for targeting foreign DNA molecules into specific sites in the plant genome using zinc finger nucleases (ZFNs). ZFNs are a new type of artificial restriction enzymes which are custom-designed to recognize and cleave specific DNA sequences, producing DSBs. However, technical difficulties in the design, assembly and analysis of ZFNs have hindered the use of this new technology for plant gene targeting. We have recently designed a set of constructs and cloning, biochemical and in-planta analysis procedures for the newly designed ZFNs. Cloning begins with de-novo assembly of the DNA-binding regions of new ZFNs from overlapping oligos containing modified helices responsible for DNA triplet recognition, and their Page 26 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 insertion between a nuclear localization signal and the FokI endonuclease domain. Following the transfer of fully assembled ZFNs into E. coli expression vectors, bacterial lysates were found to be most suitable for in-vitro digestion analysis of palindromic target sequences. An in-planta activity test was also developed to confirm the nucleic activity of ZFNs in plant cells. The assay is based on reconstruction of GUS expression following bombardment of a reporter and ZFN-expressing plasmids into mesophyll cells. Our new procedures, plasmids and assays bring us one step closer to efficient implementation of ZFN-based technology for gene targeting in plant species. ---------------------------------------------------------------------------------------------------------------------------------- T - 43. Generation of Single-Copy T-DNA Transformants by the Cre/LoxP Recombination Mediated Resolution System Sylvie De Buck, Annelies De Paepe, Ingrid Peck, Gordana Marjanac, Ann Depicker Plant Systems Biology, VIB, UGent, 9052 Gent, Belgium Transgene loci obtained after Agrobacterium tumefaciens transformation are considered to be less complex than those obtained after direct gene transfer, but integration of multiple T-DNA copies into direct or inverted repeats is fairly common. Recently, it was convincingly shown that transgenic plants with high and stable heterologous protein accumulation levels can be enriched for by screening for single-copy T-DNA transformants (De Buck et al., 2004). The aim was to evaluate a transformation system to generate efficiently single T-DNA copy transformants. Therefore, a strategy was designed to reduce the number of transgene copies by making use of the site-specific Cre/loxP recombination system. A lox-T-DNA vector containing two invertedly oriented loxP sequences located both inside and immediately adjacent to the T-DNA border ends was constructed. In the presence of the CRE recombinase, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy, regardless of the orientation and number of T-DNAs integrated at one locus. Two different strategies were followed to test the approach. Firstly, several parental plants containing multiple lox-T-DNA copies at one locus were crossed with creexpressing plants. Secondly, cre-expressing plants were retransformed with the lox-T- DNA construct. For both methods, we could demonstrate the resolvement of multimeric T-DNAs to one T-DNA copy with a satisfactory frequency. Furthermore, this reduction in T-DNA copies led in most cases to an increased transgene expression level. We therefore propose that the described lox-T-DNA vector is a tool to obtain transformants with high and stable transgene expression. Reference: De Buck, S., Windels, P., De Loose, M., and Depicker, A. (2004). Single-copy beta-glucuronidase transgenes integrated at different positions in the Arabidopsis genome show uniform and comparable expression. Cell. Mol. Life Sci., 61: 2632-2645. ---------------------------------------------------------------------------------------------------------------------------------- Page 27 sur 56
Page 1: PLANT DNA Repair & Recombination In
Page 4 and 5: EMBO Plant DNA Repair and Recombina
Page 6 and 7: 1400-1700 SESSION V: Recombination
Page 20 and 21: eing propagated. EMBO Plant DNA Rep
Page 54 and 55: Cyril Charbonnel CNRS UMR6547 Unive
Page 56 and 57: Sean Mayes University of Nottingham
Page 58: Wanda Melody Waterworth University

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