Investigators at The Scripps Research Institute (TSRI) say they have found a way to use a new DNA-editing technology more broadly than in the past. The scientific advance involves a set of designer DNA-binding proteins called TALEs (transcription-activator-like effectors), which biologists increasingly use to turn on, turn off, delete, insert, or even rewrite specific genes within cells.

The researchers see potential applications in the biotech and medical arenas including gene therapy, as well as in scientific experiments. TALE-based methods had been considered useful against only a fraction of the possible DNA sequences found in animals and plants, but the new finding removes that limitation, according to the TSRI team.

“This is one of the hottest tools in biology, and we’ve now found a way to target it to any DNA sequence,” said Carlos F. Barbas III, Ph.D., the Janet and Keith Kellogg II Chair in molecular biology and professor in the department of chemistry at TSRI. He and his group just published their study, entitled “Directed Evolution of the TALE N-Terminal Domain for Recognition of All 5’ Bases,” in an advance online edition of Nucleic Acids Research.

Scientists have found that they can easily engineer the DNA-grabbing segment of TALE proteins to bind precisely to a DNA sequence of interest. Typically they join that DNA-binding segment to another protein segment that can perform some desired function at the site of interest.

However, TALE-based DNA-editing has been seen to have a significant limitation. Virtually all the natural TALE proteins that have been discovered so far target sequences of DNA whose transcription begins with thymidine. Structural studies have hinted that natural TALE proteins can’t bind well to DNA without that initial T. Molecular biologists thus have widely assumed that the same “T restriction” rule applies to any artificial TALE protein they might engineer.

“Yet no one has investigated thoroughly whether that initial thymidine is truly required for the variety of TALE designer proteins and enzymes that now exist,” said Brian M. Lamb, Ph.D., a research associate in the Barbas Lab who was first author of the new study.

Dr. Lamb started by evaluating how well TALE-based proteins function against their usual DNA targets when the first DNA letter is switched from a T to one of the other three nucleosides (A, G, or C). Using a library of natural and engineered TALE proteins, he found strong evidence in favor of the “T restriction” rule. “There was an orders-of-magnitude difference: Some of the TALE proteins we evaluated lost as much as 99.9% of their activity when we changed that first nucleoside base,” said Dr. Lamb.

But he pointed out that he wasn’t ready to give up on the possibility of designing more broadly useful TALE proteins. For this he adapted a “directed evolution” technique developed last year by Andrew C. Mercer, Ph.D., who at the time was another research associate in the Barbas lab. First, Dr. Lamb generated a large library of novel TALE proteins that vary randomly in the structures they hypothesized to grab the initial nucleoside. He then put these new TALEs through a series of tests to select those that work adequately even with a non-T nucleoside at the start of their target DNA sequence.

In this way, he found several new TALE protein architectures that aren’t held back by the T restriction. One prefers to bind to DNA that begins not with a T nucleoside but with a G (guanosine). Others bind well enough to sequences that start with any of the four DNA nucleosides. Lamb found that these non-T-restricted TALEs work as designed when conjoined, for example, to DNA-cutting enzyme fragments. “Essentially we abolished the T requirement,” noted Dr. Lamb.

“That means that the number of DNA sites we can target with TALE-based proteins, and the precision with which we can target within any given gene, have gone up dramatically,” added Dr. Barbas.

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