Using the molecular equivalent of a guided missile, researchers have found a way to add and remove genes at specific spots in a plant's genome. This targeted approach promises to speed the development of maize and other crops with improved growth, nutritional, or biofuel potential.
Researchers typically fly blind when trying to add or modify plant genes. Gene guns and mutagenesis techniques insert or disrupt genes randomly in the plant genome, such that plant researchers must make and screen thousands of mutants in order to find one with the right change in the right place.
About 6 years ago, Dana Carroll of the University of Utah in Salt Lake City and colleagues built zinc fingers--molecules that recognize and attach to specific DNA sequences--and joined them with enzymes that slice through the DNA at that same spot to create zinc-finger nucleases. This allowed them to target a genomic region in need of modification. At the same time, they provided new DNA to be stitched into the genome at that spot.
After seeing the procedure work in Drosophila and mammalian cells, Daniel Voytas, a plant molecular biologist at the University of Minnesota, Twin Cities, and his colleagues showed in 2005 that, in principle, the technique could work in plants as well. The trick was coming up with zinc fingers for the specific DNA target. One company provides customized zinc fingers, but Voytas, J. Keith Joung of Massachusetts General Hospital in Boston, and their colleagues have instead developed a zinc-finger resource freely available to other researchers for generating the appropriate nucleases.
The researchers' first subject was the tobacco plant. They designed nucleases to home in on a tobacco gene that controls the plant's susceptibility to certain herbicides. When they exposed tobacco cells to the zinc finger nucleases, they also added a version of that gene that enhances herbicide resistance. About 2% of the treated plants survived herbicide treatment, showing that gene swaps had occurred. This percentage is high by gene-modification standards, Voytas and his colleagues report online today in Nature.
In a second paper, also published online today in Nature, Vipula Shukla and her colleagues at Dow AgroSciences in Indianapolis, Indiana, demonstrate that the zinc-finger technology works in maize as well. This time, the researchers got two modifications for the price of one. They targeted a gene called IPK1, which ties up phosphorus in a form that animals can't digest. Animals end up excreting the phosphorus, which pollutes the ground and water. The team used zinc fingers that specifically target the IPK1 gene and then added bacterial DNA that would disrupt the gene while at the same time conferring herbicide resistance to the plant. The double shot worked: The bacterial gene disrupted the IPK1 gene between 3% and 100% of the time and also made the plants resistant to herbicides, the team reports.
Experts say the new technology has potential. There have been other techniques for targeted gene modification "that seemed promising when published but that have disappeared," says David Ow, a plant molecular biologist at the U.S. Department of Agriculture in Albany, California. But "this [zinc-finger nuclease] technology is probably going to stick around," he predicts. Today, most gene modifications involve transferring genes from a different species into a crop, notes Ow, but with this technique, one can easily replace a gene with another version from the same species, potentially making it more acceptable to the public.
Agricultural biotechnology expert Roger Beachy, president of the Donald Danforth Plant Science Center in St. Louis, Missouri, is also enthusiastic. "The advance is highly significant for plant biotechnology," he says. "It brings a new level of sophistication to the [field]."