HOMOLOGOUS RECOMBINATION IN PLANTS
The various plant genome projects have made great strides in identifying the many genes that dictate plant growth and development. Understanding how plant genes work in metabolic and developmental pathways is the current challenge faced by plant functional genomics. Although a number of tools are available to study plant gene function, one tool is sorely lacking: the ability to make precise insertions, deletions or substitutions in plant genes through homologous recombination or gene targeting. Gene targeting will make it possible to take plant genomics to the next level, by linking DNA sequences to biological functions unique to plants. Furthermore, gene targeting enables the biosynthetic capacity of plants to be harnessed to produce the many products required by a growing world population, including specialty chemicals, proteins, oils and carbohydrates for food, medicine and industry.
The Voytas lab has implemented a method for gene targeting that allows specific DNA sequence changes to be introduced into plant chromosomes with high efficiency (Plant Journal 44:693). 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 or endogenous 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 replacement and robust methods for the design of zinc finger arrays. Achieving this goal is facilitated by the Zinc Finger Consortium—a group of scientists dedicated to promoting applications of zinc finger proteins for genome modification. We anticipate the outcome of our research program will be a highly facile gene targeting system that can be employed in a variety of plant species to study gene function.