Howard Hughes Medical Institute (HHMI) scientists have created a portrait of a DNA-snipping protein called Cas9, a powerful research tool used in many labs for genome editing.

Howard Hughes Medical Institute (HHMI) scientists have created a portrait of a DNA-snipping protein called Cas9 that reveals how the enzyme reshapes itself when it associates with an RNA molecule, creating a channel that lets it find and cleave its DNA target. The protein-RNA complex is a key component of a primitive bacterial immune system called CRISPR, and is also being used by researchers to cut specific DNA sequences in the laboratory for genome editing applications.

“These proteins have been wildly successful at inducing site-specific genetic changes in virtually every system in which they've been tried,” says HHMI Investigator Jennifer A. Doudna, one of the leaders of the study. The new understanding of the structural transformations that Cas9 undergoes as it first encounters an RNA guide—and later, DNA—helps explain how the enzyme works and offers insight into how researchers can control its activity, she says.

Doudna, who is at the University of California, Berkeley, led the structural and biochemical analyses of Cas9 together with HHMI Investigator Eva Nogales at the Lawrence Berkeley National Laboratory and Emmanuelle Charpentier at Helmholtz-Zentrum für Infektionsforschung GmbH in Germany. Their work was published February 7, 2014, in the journal Science

Many bacteria have a CRISPR-based immune system that is used to recognize and destroy the genomes of invading viruses and plasmids. In 2012, the labs of Doudna and Charpentier showed that Cas9 is a DNA-cutting enzyme guided by RNA. Cas9 relies on two short RNA guide sequences to find foreign DNA, then cleaves the target sequences, thereby silencing the invaders' genes.

The process is specific and efficient enough to fend off viral infections in bacteria, and those same qualities have made the CRISPR system a powerful research tool. Doudna's team adapted the system so that it can be guided by a single short RNA molecule. Researchers who use the system for genome editing can customize that RNA so that it directs Cas9 to cleave at a desired location in the genome.

For the current study, Doudna, Nogales, and Charpentier teamed up to get a closer look at the enzyme. “We are very keen to understand how this enzyme is able to recognize DNA as specifically as it does, and what controls its activity in cells,” Doudna says. The team chose to look at two versions of Cas9, taken from different species of bacteria. One protein is about 25 percent larger than the other, but both cleave DNA targets as directed by RNA.

Martin Jinek, an HHMI research specialist in Doudna's lab who now directs his own laboratory at the University of Zurich, and Fuguo Jiang, a postdoctoral researcher in Doudna's lab, used x-ray crystallography to determine the detailed atomic structure of each of the two Cas9 proteins. The most widely used method for structure determination is x-ray crystallography. In that technique, crystallized proteins are first bombarded with x-ray beams. As the x-rays bounce off of atoms in the crystal, they leave a diffraction pattern, which can then be analyzed to determine the three-dimensional shape of the protein.

When they studied the structure of Cas9, the researchers found it was made of two lobes, with the DNA-cleaving regions clustering together in a single lobe. The variable regions of the proteins fell within the second lobe, which accounted for their size differences. Further experiments are needed to test how the variations in the second lobe affect protein function, but Doudna says the findings are encouraging for genome editing applications.

“A lot of people are interested in whether this Cas9 enzyme can be engineered to be even smaller than any of the natural variants,” she says, explaining that some of the vectors used to introduce Cas9 into cells tolerate only small inserts. “Based on comparing these two structures, we think that evolution has kind of already done this: It's already chewed away at one lobe of the protein, which is away from where the catalytic sites are. We also think that it may be possible to do even more trimming and generate a protein that retains all the functionality of Cas9, but is in a more streamlined state.”

The x-ray crystallography studies offered the researchers a detailed view of the Cas9 protein alone. But they were also curious how the globular enzyme interacted with RNA and DNA to find its target.

They chose to visualize Cas9 using electron microscopy, a technique that can provide as much structural detail as x-ray crystallography, but also allows researchers to look at proteins in solution, in which they can interact naturally with other molecules. Samuel Sternberg, a graduate student in Doudna's lab, and David Taylor, a joint postdoctoral researcher in Nogales' and Doudna’s labs, used electron microscopy to capture images of Cas9 alone, then in association with an RNA guide molecule, and finally interacting with DNA.

What they found, Nogales explains, is that when Cas9 associates with RNA, it reconfigures, forming a channel in the center of the protein—preparing itself, it seems, to interact with DNA. Indeed, when the Cas9-RNA complex was allowed to interact with DNA, the researchers saw tips of the DNA molecule—to which they had attached protein tags for visualization—protruding from the ends of the channel. With additional biochemical experiments, they also showed how loops in the Cas9 protein contribute to DNA recognition, enabling Cas9 to interact specifically with small DNA motifs that Doudna and colleagues have recently shown enable Cas9 to begin scanning in search of its target sequence.

Together, these findings provide a comprehensive view of Cas9's structure and conformational changes during complex assembly, the scientists say. “Unexpectedly, the guide RNA, which was thought to serve basically as the template for determining what DNA is going to be cleaved, is most likely required to activate the protein by changing its conformation,” says Nogales. Researchers will need to keep this function in mind if they intend to simplify the CRISPR system for biotechnology applications, she adds. “This is a minimal requirement that cannot be forgotten.”

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