R-loop formation and conformational activation mechanisms of Cas9

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage(1-)(3). The programmable activity of Cas9 has been widely utilized for genome editing applications(4-6), yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcuspyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodatesthe distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

Automated summary

Researchers have used cryo-electron microscopy to observe the structural changes that occur during the binding and activation of Cas9. These findings provide a valuable structural framework for understanding the conformational checkpoint mechanism of Cas9 and may guide the development of new Cas9 variants and guide RNA designs with enhanced specificity and activity.