Searching a whole genome for one particular sequence is like trying to fish a specific piece from the box of a billion-piece puzzle. Using advanced imaging techniques, Univ. of Illinois researchers have observed how one set of genome-editing proteins finds its specific targets, which could help them design better gene therapies to treat disease.
Illinois chemical and biomolecular engineering professors Charles Schroeder and Huimin Zhao, along with graduate students Luke Cuculis and Zhanar Abil, published their work in Nature Communications.
TALE proteins, or transcription activator-like effectors, can be programmed to recognize and bind to specific regions of DNA. Researchers have been interested in using TALE proteins for synthetic biology, such as genome editing in plants or bacteria, or for gene therapy. For example, Zhao’s group explores using TALE proteins to treat sickle cell anemia, which is caused by a mutation in one link of the DNA chain.
“People have been using this technique, but nobody fully understood the mechanism before,” Schroeder said. “The main question is, how do these proteins find their target sites? They are designed to bind to a particular site, but there’s this big genome with billions of bases, so how does the protein find its site? If you understand the mechanism, you might be able to engineer better, more improved proteins.”
The researchers used imaging techniques that let them watch through a microscope how individual TALE proteins interact with a string of DNA. They observed that the proteins seek and find using a combination of sliding and hopping. The proteins bind to the DNA and slide along the helix, traveling down the DNA molecule like a highway. The researchers also observed that the proteins perform frequent, short hops along their paths, allowing them to move more efficiently but never straying far from the DNA.
“The combination of sliding and hopping means they can cover more ground and potentially move past obstacles that might be in their way,” said Cuculis, a co-first author of the paper.
“The combination of behaviors also would allow a TALE protein to switch strands of the DNA double helix and sample both strands, increasing the chance of finding its target site,” said Abil, the other co-first author.
They also analyzed which parts of the protein did the work, finding a division of labor among the domains within the protein: One part searches, while another part binds to the specific target sequence. This finding excited the researchers, because it gave them insight into where to tweak the protein design so that it binds even more selectively.
“The major goal would be to engineer improved proteins that have lower off-target binding. You don’t want them to bind to the wrong place,” Schroeder said. “If we engineer a protein in such a way where we don’t just naively change the specific binding domain, but design a new protein with distinct parts of the protein in different places, we might be able to increase the efficiency without increasing mistakes.”
Next, the researchers are watching the proteins work in live cells to see if the behavior changes when immersed in the bustling activity within the cell nucleus.
“It gives us a better understanding of the genome editing mechanism,” Zhao said. “When we understand it better, it provides new insights for the design of the protein. If you really talk about therapeutic applications, it needs to be a specific design.”