Two teams worked closely to reveal the structural details of the Cas9 complex and to test their functional significance. Their efforts revealed a division of labor within the Cas9 complex. The researchers determined that the Cas9 protein consists of two lobes: One lobe is involved in the recognition of the RNA and DNA elements, while the other lobe is responsible for cleaving the target DNA, causing what is known as a “double strand break” that disables the targeted gene. The team also found that key structures on Cas9 interface with the guide RNA, allowing Cas9 to organize itself around the RNA and the target DNA as it prepares to cut the strands.
Identifying the key features of the Cas9 complex should enable researchers to improve the genome-editing tool to better suit their needs.
“Up until now, it has been very difficult to rationally engineer Cas9. Now that we have this structural information, we can take a principled approach to engineering the protein to make it more effective,” says Zhang, who is also a co-founder of Editas Medicine, a company that was started last year to develop Cas9 and other genome-editing technologies into a novel class of human therapeutics.
The potential is there for bacteria and other microbes to be genetically engineered to perform a cornucopia of valuable goods and services, from the production of safer, more effective medicines and clean, green, sustainable fuels, to the clean-up and restoration of our air, water and land. Cells from eukaryotic organisms can also be modified for research or to fight disease.
An international collaboration that used x-ray crystallography to produce 2.6 and 2.2 angstrom (Å) resolution crystal structure images of two major types of Cas9 enzymes. The collaboration then used single-particle electron microscopy to reveal how Cas9 partners with its guide RNA to interact with target DNA. The results point the way to the rational design of new and improved versions of Cas9 enzymes for basic research and genetic engineering.
The crystal structure of SpyCas9 features a nuclease domain lobe (red) and an alpha-helical lobe (gray) each with a nucleic acid binding cleft that becomes functionalized when Cas9 binds to guide RNA.
“The combination of x-ray protein crystallography and electron microscopy single-particle analysis showed us something that was not anticipated,” says Nogales. “The Cas9 protein, on its own, exists in an inactive state, but upon binding to the guide RNA, the Cas9 protein undergoes a radical change in its three-dimensional structure that enables it to engage with the target DNA.”
“Because we now have high-resolution structures of the two major types of Cas9 proteins, we can start to see how this family of bacterial enzymes has evolved,” Doudna says. “We see that the two structures are quite different from each other outside of their catalytic domains, suggesting an interesting structural plasticity that could explain how Cas9 is able to use different kinds of guide RNAs. Also, the differences in the two structures suggest that it may be possible to engineer smaller Cas9 variants and still retain function, an important goal for some genome engineering applications.”
Currently, Cas9 is used in experiments to silence genes in mammalian cells — sometimes at multiple sites across the genome — and large libraries of RNA sequences have been created to guide Cas9 to genes of interest. However, the system can only target specific types of sites. Some studies have also shown that the RNA could lead Cas9 “off target,” potentially causing unexpected problems within the cellular machinery.
The researchers plan to use this new, detailed picture of the Cas9 complex to address these concerns.
“Understanding this structure may help us engineer around the current limitations of the Cas9 complex,” says co-author F. Ann Ran, a graduate student in Zhang’s lab. “In the future, it could allow us to design versions of these editing tools that are more specific to our research needs. We may even be able to alter the type of nucleic acid sequences that Cas9 can target.
SOURCES – MIT, Lawrence Berkeley National Lab