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[资源] 【推荐】Plants genes get fine tailoring

Technique allows plant researchers to target and replace specific genes.

After decades of searching, plant biologists have found a way to selectively snip out one gene and replace it with another. The method promises to be a boon to both basic research and the creation of genetically engineered crops, observers say.

The technique relies on enzymes called zinc-finger nucleases, which bind to specific sites in a genome and then cut nearby strands of DNA. When the cell repairs the cut DNA, the gap can be either simply sealed — in effect deleting the targeted gene — or filled in with a new gene. Zinc-finger nucleases have recently been used to create human immune cells that are resistant to HIV (see 'Designer protein tackles HIV').

Now, that technique has been expanded to include plants. In papers published online today by Nature, two independent groups of researchers report that the technique can also be used to engineer herbicide-resistant corn and tobacco1,2.

"It's a great achievement," says David Ow, a plant biologist at the US Department of Agriculture Plant Gene Expression Center in Albany, California. "The fact that two groups have succeeded is very promising."

A troubled history
Plant biologists have long been frustrated by the lack of a simple method for either deleting a specific gene from the genome or replacing it with another gene. Even Arabidopsis thaliana, the fast-growing weed with a small genome favoured by many plant biologists as a model system, has not been amenable to targeted gene replacement. "To have a really good model system you need targeted gene replacement," says Joseph Ecker, a plant biologist at the Salk Institute for Biological Studies in La Jolla, California. "We've been kind of limping along without it."

Sporadic reports of plant gene-replacement strategies have come and gone, but none has been versatile or efficient enough for wide-scale use. In 1997, a Nature paper reporting targeted gene disruption in Arabidopsis raised the hopes of many plant researchers3. "When that paper came out, we all thought 'This is it,'" says Ow. "Unfortunately it didn't pan out. The frequency [of success] was very low."

One problem is that plants tend to have big, complex genomes, chock full of large families of genes with very similar DNA sequences, says Vipula Shukla, a scientific group leader at Dow AgroSciences in Indianapolis, Indiana. That makes targeting a specific gene more difficult. "The challenges associated with any kind of sequence-specific modification in plants are profound," she says.

For one of the new studies, Shukla and her colleagues at Dow AgroSciences teamed up with Sangamo BioSciences, a company based in Richmond, California, that has developed a proprietary method for engineering zinc-finger nucleases2. The team has used zinc fingers to replace a gene called IPK1 with an herbicide-resistance gene.

Meanwhile, the other study1 is the work of a research team led by Daniel Voytas, a plant biologist at the University of Minnesota in Minneapolis and a member of the Zinc Finger Consortium, a group of academic researchers united to develop open-access zinc-finger technology (see 'The fate of fingers'). Voytas's group has engineered herbicide-resistant tobacco by inserting specific mutations into a gene called SuR.

Effective but costly
Both groups have replaced their selected genes at a frequency much higher than anyone has achieved before, says Ow. But some technical hurdles could remain. For instance, designing zinc fingers that target only one gene will probably still be a challenge, says Holger Puchta, a plant biologist at Karlsruhe University in Germany who is developing zinc-finger nucleases for use in Arabidopsis. "Many zinc-finger nucleases also cut other sites with similar sequences," he says. "This is still a big problem in all organisms."

Shukla notes that her team was able to target IPK1 without affecting a 98%-identical gene called IPK2. Voytas' team was also able to target their gene without hitting another gene that is 96% identical. But Voytas adds that some of the zinc-finger nucleases the team studied did cleave both genes, even after preliminary studies in cell cultures suggested the nucleases were specific

Dow AgroSciences may collaborate with another company to make its technology platform available to plant researchers, but pricing has not been determined. Meanwhile, Keith Joung, a member of the Zinc Finger Consortium and a researcher at Harvard Medical School in Boston, says that zinc-fingers cost his team about $1,000 to make. But his laboratory has extensive experience with the technique, Young says, and costs could be higher for other labs.

The technique could also assuage a common concern about transgenic crops. "One argument that is often used — in part correctly — is that when we create transgenic plants, we insert the transgene somewhere in the genome, and we don't know exactly where it happens to insert," says Wilhelm Gruissem, a plant biologist at the Swiss Federal Institute of Technology in Zurich. "Now you can target the transgene to a specific location."

References
Townsend, J. A. et al. Nature doi:10.1038/nature07845 (2009).
Shukla, V. K. et al. Nature doi:10.1038/nature07992 (2009).
Kempin, S. A. et al. Nature 389, 802–803 (1997).

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