Choose your own splicer

Freely available tool aids genetic research and gene therapy

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Think of them as Swiss Army knives for DNA.

Zinc-finger proteins can cut, splice or tweak a targeted gene, and a new “open source” method for making customized zinc-finger proteins aimed at specific genes will give scientists easier access to this powerful genetic tool.

“There’s tremendous potential for the technology,” both for biology research and for altering a person’s DNA to treat genetic diseases such as sickle cell anemia, says lead scientist J. Keith Joung, a protein engineering expert at Harvard Medical School and Massachusetts General Hospital in Boston.

Named for the zinc ions that hold the proteins together, zinc-finger proteins latch on to DNA at specific locations. Each variety of zinc-finger protein targets a unique sequence of genetic code, and scientists can couple these proteins with enzymes that then cleave the DNA at that spot or alter the DNA in other ways.

Despite the proteins’ potential, progress in the nascent field has been impeded by lack of access to customization tools, Joung says.

“Giving academics the ability to practice with it will move the field forward faster,” Joung says. The research appears in the July 25 Molecular Cell.

“I feel they’re going to be very powerful tools to modify the genome,” comments Carlos Barbas, a zinc-finger protein expert at the Scripps Research Institute in La Jolla, Calif. Barbas had previously posted his methods for making zinc-finger proteins on his lab’s website.

To target a gene — perhaps one that causes a disease — a scientist must first make customized zinc-finger proteins that bind to the specific sequence of DNA “letters” that make up that gene’s genetic code.

Other methods for customizing these proteins are publicly available, but they have drawbacks. Existing techniques are either difficult for non-experts to perform, or the proteins they produce aren’t strongly targeted to the desired gene, Joung says.

Access to a relatively easy, effective technique has thus far been limited. “The reason that that has been an issue in our field is because of this company called Sangamo,” Joung says.

Sangamo BioSciences, Inc. of Richmond, Calif., has positioned itself as the Microsoft of zinc-finger proteins. “They have licensed pretty much all of the underlying technology,” comments Dana Carroll, a zinc-finger protein expert at the University of Utah School of Medicine in Salt Lake City. “Other people can play the game if they play with Sangamo’s ball.”

While the company recently made a deal with Sigma-Aldrich, a biology research supply company based in St. Louis, to provide their proprietary zinc-finger proteins to researchers, scientists outside of Sangamo still won’t have the tools to make small adjustments to the proteins needed for their experiments.

The new, freely available technique is “a method for engineering zinc-finger proteins where everything is transparent,” Joung says. “If you practice it, you know how everything is occurring.”

“I am delighted to see the … new method for rapid ‘open source’ engineering of [zinc-finger proteins],” comments Srinivasan Chandrasegaran, who led the team at Johns Hopkins University in Baltimore that pioneered the field in 1996 by making the first DNA “scissors” called zinc-finger nucleases. “It should provide academic researchers with easy access to custom zinc-finger nucleases, allowing them to address various biological and biomedical problems.”

To make customized zinc-finger proteins, Joung’s team drew from 66 pools of proteins. Each pool contained proteins targeted to bind with a certain three-letter “word” in the genetic code. A custom zinc-finger protein must string together individual proteins from three of these pools, so that the finished zinc-finger protein targets a total of nine letters of DNA.

The tricky part is that, when the individual proteins are strung together, they can interfere with each other. So even if each protein on its own binds strongly to its three-letter word, it might bind much more weakly when assembled into a complete zinc-finger protein. One possible outcome is that the zinc-finger protein could also bind to — and hence cut, split or tweak — other, unintended genes.

So the new technique includes a screening step. After making thousands of candidate zinc-finger proteins, the scientists engineered the bacterium E. coli so that when a zinc-finger protein binds to the desired nine-letter sequence of genetic code, it causes a marker protein to be produced. The researchers then screened colonies of the bacteria for individuals who had the highest amounts of this marker protein. The test thus highlighted which zinc-finger protein worked best.

Joung says his team plans to make the pools of targeted proteins available to academic researchers for “some nominal fee to recover costs.” They will also make step-by-step protocols for the screening process freely available.

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