Phage-assisted continuous evolution has been adapted to the development of TALENs with better DNA-binding activity and specificity. [iStock/Blablo101]
Phage-assisted continuous evolution has been adapted to the development of TALENs with better DNA-binding activity and specificity. [iStock/Blablo101]

Before genome editing can be used more widely for clinical purposes, researchers need to develop better-targeted DNA-binding proteins. Even though existing genome-editing systems are nearly always “on target,” off-target effects are still a concern—problems caused by off-target edits could be at least as serious as problems resolved by on-target edits.

The difficulty, however, is developing better-targeted genome-editing systems quickly enough. But help is at hand, and from an unlikely source: the bacteriophage. In the laboratory of David R. Liu at Harvard University, a method has been developed that allows bacteriophages to rapidly evolve DNA-binding proteins autonomously so that they become more targetable over time.

Originally, the method, called phage-assisted continuous evolution (PACE), was used to rapidly evolve RNA polymerases and proteases with tailor-made properties. But now it has been used to continuously evolve DNA-binding domains called TALENs (transcription activator-like effector nucleases) with altered or improved DNA-binding specificity.

The new work appeared August 10 in Nature Methods, in an article entitled, “Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.”

“Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity,” wrote the authors. “DB-PACE brings the power of continuous evolution to bear on improving the activity and specificity of a variety of DNA-binding proteins.

“Because DB-PACE does not require the use of targeted libraries that can constrain or bias evolutionary outcomes, it naturally supports the discovery of evolved solutions with desired properties that could not be rationalized a priori. Furthermore, DB-PACE coupled with in vitro specificity profiling represents a new systematic approach to removing specific off-target activities of TALENs, and it may be used to facilitate generation of highly specific genome engineering tools for therapeutic applications.

The study’s lead authors, Basil Hubbard, Ph.D., an assistant professor of pharmacology at the University of Alberta, characterized DB-PACE as follows: “This technology allows you to systematically say, 'I want to target this DNA sequence, and I don't want to target these others,' and it basically evolves a protein to do just that. Using this system, we can produce gene editing tools that are 100 times more specific for their target sequence.”

Currently, much of the research exploring clinical applications of genome engineering is focused on treating monogenic diseases—diseases that involve a single gene—as they're much easier for researchers to successfully target. Examples include diseases such as hemophilia, sickle cell anemia, muscular dystrophy and cystic fibrosis. Conceivably, the problems introduced by off-target effects could become increasingly intractable as more genes are targeted simultaneously.

While the field is still in its relative infancy, Dr. Hubbard said human clinical trials involving sequence-specific DNA-editing agents are already underway. If successful, he expects the first clinical applications could be seen in the next decade. He hopes his current work will play a role in helping genome engineering reach its full potential and plans to continue his research in the quickly expanding field.

“Whereas traditional pharmaceutical drugs have a transient effect, gene editing could possibly provide a permanent cure for a lot of different diseases,” added Dr. Hubbard. “We still have to overcome many hurdles, but I think this technology definitely has the potential to be transformative in medicine.”

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