Doudna, Nogales and their collaborators addressed this knowledge deficit by first solving the three-dimensional crystal structures of two Cas9 proteins, representing large and small versions, from Streptococcus pyogenes (SpyCas) and Actinomyces naeslundii (AnaCas9) respectively. Using protein crystallography beamlines at Berkeley Lab's Advanced Light Source and the Paul Scherer Institute's Swiss Light Source, the collaboration discovered that despite significant differences outside of their catalytic domains, all members of the Cas9 family share the same structural core. The high resolution images showed this core to feature a clam-shaped architecture with two major lobes - a nuclease domain lobe and an alpha-helical lobe. Both lobes contained conserved clefts that become functional in nucleic acid binding.
"Our understanding of Cas9's structure was not complete with only the x-ray data because the protein in the crystals had been trapped in a state without its associated guide RNA," says Sam Sternberg, a member of Doudna's research group and a co-author of the Science paper. "Understanding how RNA-guided Cas9 targets matching DNA sequences for genome engineering and how this reaction and its specificity might be improved required an understanding of how the shape of Cas9 changes when it interacts with guide RNA, and when a matching DNA target sequence is bound."
The collaboration employed negative-staining electron microscopy to visualize the Cas9 protein bound to either guide RNA, or both RNA and target DNA. The structures revealed that the guide RNA binding structurally activates Cas9 by creating a channel between the two main lobes of the protein that functions as the DNA-binding interface.
"Our single particle electron microsco
|Contact: Lynn Yarris|
DOE/Lawrence Berkeley National Laboratory