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EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 ---------------------------------------------------------------------------------------------------------------------------------- P - 34. Towards efficient gene targeting in plants Seiichi Toki, Masaki Endo, Ryuya Ohishi, Masaaki Umeda, Kiyomi Abe, Hiroaki Saika, Keishi Osakabe. Plant Genetic Engineering Research Unit, National Institute of Agrobiological Scienced, Ibaraki 305-8602 Tsukuba, Japan Modification of genes by gene targeting (GT) should be the most precise gene manipulation technique and direct method for future molecular breeding in plants. In order to improve the frequency of GT, we are currently applying several approaches including the use of the zinc-finger nucleases, chromatin remodeling factors and cell cycle regulators. We are also trying to establish the model gene targeting system in Arabidopsis and rice. In this paper we would like to present current status of our study and discuss potential use of our study for the practical molecular breeding in plants. ---------------------------------------------------------------------------------------------------------------------------------- P - 35. High-frequency site directed modification of the tobacco SuRB locus mediated by zinc-finger nucleases Jeffrey A. Townsend, David A. Wright, Ronnie J. Winfrey, Stacey Thibodeau-Beganny, Magdalena Eichtinger, J. Keith Joung, Daniel F. Voytas. Genetics, Cell and Developmental Biology, Iowa State University, 50011-3650 Ames, Iowa, United States Zinc-finger (ZF) proteins can be engineered to recognize unique chromosomal sites. When fused to a nuclease domain they make targeted double-strand breaks that stimulate homologous recombination between the chromosome and an extrachromosomal DNA donor. Here we demonstrate high frequency, site directed modification of the tobacco SuRB locus using engineered zinc-finger nucleases. The SuRB gene codes for acetohydroxyacid synthase (AHAS) the target enzyme for sulfonylurea and imidazolinone herbicides. Single amino acid changes in the enzyme confer herbicide resistance. Substitutions at amino acid P196A and S647T confer sulfonylurea and imidazolinone resistance respectively, while a W573L substitution confers resistance to both herbicide classes. Promotorless, nonfunctional fragments of the SuRB gene that encode these substitutions plus a novel restriction site have been cloned and introduced into tobacco protoplasts with pairs of zinc-finger nucleases designed to recognize specific SuRB sequence. The ZF proteins were generated by selection using a bacterial cell-based twohybrid (B2H) scheme. Herbicide resistant colonies occurred at a rate of more than 3% of that given by the control, functional SuRB mutants (3% of illegitimate recombination). The herbicide resistance profile of the recombinants in all cases matches that expected given the donor DNA used. ---------------------------------------------------------------------------------------------------------------------------------- P - 36. Designed zinc finger nucleases as genome editing tools Fyodor Urnov, Jeffrey Miller, Russell DeKelver, Erica Moehle, Jeremy Rock, Kenneth Kim, Edward Rebar, Philip Gregory, Michael Holmes. Genomics, Sangamo BioSciences, 94804 Richmond, United States Designed zinc finger nucleases (ZFNs) provide a generally applicable solution to a fundamental problem in biology: the efficient alteration of genotype in plant and animal cells to an investigator-specified allelic form. Work by Jasin et al on the homing endonuclease I-SceI (1) and the invention of ZFNs by Chandrasegaran et al (2) provided the basis for work by Carroll et al on ZFN-driven homologous recombination in Xenopus (3), followed by studies of Porteus and Baltimore on reporter gene targeting in human cells (4), which, importantly, have been recapitulated in Arabidopsis by Drews et al (5) and in tobacco cells by Voytas et al (6). The transition to editing native loci of one’s choosing was enabled by the development of designed zinc finger proteins (7) that allow Page 46 sur 56
EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 one to target a DSB to essentially any position in the genome of any cell, and yield rapid, selection-free, high efficiency gene correction (8) and gene addition (9) at endogenous loci. We will describe recent progress in designed ZFN technology: our development of massively parallel tools for studying genome editing, the structure-based redesign of ZFNs aimed at ensuring single-locus specificity of the editing process, and the remarkable efficiency of single-step endogenous gene disruption we have observed in a broad range of cell types. 1. Rouet, P., et al. 1994. Proc Natl Acad Sci U S A 91:6064-8. 2. Kim, Y. G., et al. 1996. Proc Natl Acad Sci U S A 93:1156-60. 3. Bibikova, M., et al. 2001. Mol Cell Biol 21:289-97. 4. Porteus, M. H., and D. Baltimore. 2003. Science 300:763. 5. Lloyd, A., et al. 2005. Proc Natl Acad Sci U S A 102:2232-7. 6. Wright, D. A., et al. 2005. Plant J 44:693- 705. 7. Pabo, C. O., et al. 2001. Annu Rev Biochem 70:313-340. 8. Urnov, F. D., et al. 2005. Nature 435:646-51. 9. Moehle, E. A., et al. 2007. Proc Natl Acad Sci U S A 104:3055-3060. ---------------------------------------------------------------------------------------------------------------------------------- P - 37. Strategies to modify and monitor gene targeting and meiotic recombination in crops. Philippe Vain, Vera Thole, Barbara Worland, Katie Mayes, Upendra Devisetty, Chanate Malumpong, Sean Mayes. Agricultural and Environmental Sci (Biosciences), University of Nottingham, LE12 5RD Loughborough, United Kingdom Significant progress has been made recently in perturbing genetic recombination processes in model plants, particularly Arabidopsis thaliana and tobacco. While these approaches have not yet coalesced into practical and efficient methods to achieve Gene Targeting (GT) and to modify meiotic recombination, a firm foundation is being laid. We have been looking at opportunities to test promising results in model di- and monocotyledonous species, with an ultimate aim to manipulate these processes in crop species, particularly wheat. We have developed a deficient GUS-based marker system, which could be repaired by GT. As this system does not require plant regeneration, we can rapidly examine very large numbers of events. A number of genes involved in recombination, particularly in rice have been cloned into expression vectors and we are beginning to characterise the effect of these genes in A. thaliana as a first step. The system we have developed should allow the same recombination genes to be tested in a number of species (A. thaliana, tobacco and rice), allowing rapid follow up of positive results from models to crops. We will use the same transgenic lines that have been tested for GT effects to assess effects in meiotic recombination, through crossing and microsatellite analysis. In addition to this, we are currently screening neutron deletion material of wheat to identify deletion of specific homeologues of RAD51 and DMC1 (which may not cause full sterility in this hexaploid) and intend to compare these with characterisation of rice Tos17 insertional inactivation lines for the rice equivalents (where two versions of each gene exist). Results to date and our future plans will be presented. ---------------------------------------------------------------------------------------------------------------------------------- P - 38. Reverse Progeny Mapping Aat Vogelaart, Johan Schut, Rob Dirks, Cilia Lelivelt. Celbiology, Rijk Zwaan Breeding BV, 4793 RS Fijnaart, Netherlands A method is provided for mapping traits in organisms, in particular in plants. The method comprises a) providing a population of SDR-0 organisms, that each arise from one member of a population of unreduced cells resulting from second division restitution, in particular a population of unreduced spores; b) producing SDR-I progeny populations of each of these SDR-0 organisms; c) phenotyping the SDR-I progeny populations to identify segregating traits within each SDR-I progeny population; d) if segregating progeny is present in a SDR-I progeny population genotyping the corresponding SDR-0 organism and comparing the genotype thereof with the genotype of the other SDR-0 organism to identify heterozygous chromosomal regions associated with Page 47 sur 56
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