A Homologous Recombination System for Plants Based on Zinc Finger Nucleases

Iowa State University, Ames IA

Investigators

Abstract

Plants have remarkable biosynthetic capacities that can be harnessed to produce a vast array of complex molecules. The next generation of crops will synthesize specialty chemicals and proteins of value for food, medicine and industry. Fully harnessing the biosynthetic potential of plants, however, requires understanding the genetic basis by which plants control growth and development and respond to environmental signals and stresses. Once an understanding of plant gene function is attained, it will be possible to modify plant genomes to create crops that meet the burgeoning need for plant-derived products. Discerning plant gene function and re-orchestrating the plant genetic code require sophisticated tools to manipulate plant genomes. Specifically, it would be desirable to make a variety of alterations to the plant genetic code, including precise DNA insertions, deletions or substitutions. Gene targeting or homologous recombination is a method that allows specific DNA sequence changes to be introduced into plant chromosomes. 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 any site in the plant genome, thereby making it possible modify any chromosomal sequence. Current research is directed at developing zinc finger nuclease-assisted gene targeting for widespread use, including establishing key parameters for high frequency gene targeting and robust methods for the design of zinc finger arrays. The outcome of this research project will be a highly facile gene targeting system that can be employed in a variety of plant species to study gene function. The gene targeting platform will also enable the engineering of crop plants with novel traits, including those that better withstand pests, have enhanced food value, and produce compounds of industrial importance. 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. This may reduce barriers imposed on genetically modified crops and thereby increase the value of U.S. commodities in a global marketplace.