The researchers’ findings were published February 6 in Science, in an article entitled “Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.” In this article, the authors observed that the “architectures of Cas9 enzymes define nucleic acid binding clefts, and the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound.”
Despite significant differences outside of their catalytic domains, all members of the Cas9 family share the same structural core. This core is shaped rather like a clam. Instead of paired shells, the core has its two cleft-harboring lobes—a nuclease domain lobe and an alpha-helical lobe. Upon binding with guide RNA, the two lobes reorient so that the two clefts face each other, forming a central channel that interfaces with target DNA.
The authors emphasized that the identification of variable regions appended to a conserved Cas9 structural core provides a rationale for the diversity of crRNA:tracrRNA guide structures recognized by Cas9 enzymes. Also, these details suggest a framework for protein engineering approaches aimed at altering catalytic function, guide RNA specificity, or protospacer adjacent motif (PAM) requirements in targets.
Clarifying the point, Doudna said, “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.”
The Cas9 results are of particular relevance to Type II CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems. These systems, which use Cas9 to generate double-strand breaks in DNA, are part of an adaptive bacterial immune response. They have also been harnessed as a gene editing tool in many eukaryotic organisms.
For more on CRISPRs, be sure to check out our webinar "CRISPRs: Ushering in a New Age of Gene Editing".