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Precise Engineering of Plant Genomes Using Zinc Finger Nucleases

PI and Co-PIs: Daniel F. Voytas, University of Minnesota; Drena L. Dobbs, Iowa State University; J. Keith Joung, Massachusetts General Hospital; Jennifer Kuzma, University of Minnesota; Kan Wang, Iowa State University

Project abstract. Plants have remarkable biosynthetic capacities that can be harnessed to produce compounds of value for food, fuel, medicine and industry. Fully realizing the biosynthetic potential of plants, however, requires sophisticated tools to manipulate plant genomes. Specifically, it is desirable to make precise alterations to the plant genetic code, including DNA insertions, deletions and substitutions. Such precise modifications can be made through a process known as gene targeting or homologous recombination. Fundamentally, gene targeting is a DNA swapping reaction: a DNA fragment carrying a desired sequence is introduced into a plant cell, and it replaces the native copy of the gene. To enhance the efficiency of gene targeting, a chromosome break is created at the site of modification (the target). An enzyme called a zinc finger nuclease (ZFN) is used to generate the chromosome break. ZFNs have two components: a DNA recognition domain (a zinc finger array) and a nuclease that cleaves the chromosome. Zinc finger arrays can be designed to recognize diverse DNA sequences, thereby making it possible modify any chromosomal target. Current research is directed at developing zinc finger nuclease-assisted gene targeting for widespread use in plants, including establishing key parameters for high frequency gene modification and robust methods for the design of zinc finger arrays. The project focuses on implementing gene targeting in rice - the world,s most important food crop; however, the outcome of the research will be a highly facile gene targeting system that can be employed in a variety of plant species. Because gene targeting introduces changes in plant genomes in a highly specific and controlled manner, crops generated through gene targeting may be met with greater public acceptance than traditional genetically modified crops. The societal impact of 'recombinant' plants (i.e. those derived from gene targeting) will be addressed by exploring legal, policy, intellectual property, economic, social, and ethical issues surrounding this new technology.

Broader Impacts. An efficient method for making precise modifications to plant genomes (gene targeting) is critical for detailed functional analysis of genes and genetic pathways. Gene targeting will also enable the development of new crop varieties, including those that better withstand pests, have enhanced food value, and produce compounds of industrial importance. Gene targeting differs fundamentally from transgenesis in that the resulting plant material may only have a single or few nucleotide changes that distinguish it from the parent. This precision suggests that gene targeting may mitigate some concerns about the use of genetically modified crops, which has limited the application of genetic engineering to plant agriculture. The project specifically explores the potential societal impacts of gene targeting. In addition, the project will train undergraduate and graduate students for work in plant molecular biology, computational biology and public policy. This diversity of research topics provides a rich interdisciplinary training environment and a unique opportunity for all project participants to learn about the impact of science on society. Access to software and data generated from this project can be obtained at www.zincfingers.org; DNA reagents are available at www.addgene.org.