Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

TLDR

Type II CRISPR‑Cas systems use an RNA‑guided DNA endonuclease, Cas9, to generate double‑strand breaks in invasive DNA during bacterial adaptive immunity, and Cas9 has become a powerful tool for genome editing and gene regulation in eukaryotes. The crystal structures reveal that Cas9 enzymes possess nucleic acid binding clefts, and electron microscopy shows that guide RNA induces reorientation of the two structural lobes to form a central channel for DNA binding. High‑resolution structures of two Cas9 subtypes show a shared structural core, and the extensive conformational rearrangements preceding DNA duplex binding implicate guide RNA loading as a key activation step.

Abstract

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.