Cas9 nucleases of CRISPR-Cas adaptive immune systems are programmable RNA-guided DNA endonucleases that evolved from the transposon-associated IscB enzymes. Comparative structural studies have revealed substantial architectural elaboration during this evolutionary transition, including the replacement of the larger ωRNA scaffold of IscB with the REC domains of Cas9, capable of recognizing longer guide-target heteroduplexes. However, these evolutionary insights remain restricted to static structural features, and the dynamic properties that underlie nuclease activation have not been systematically explored across the IscB-to-Cas9 lineage. Here, by combining cryo-electron microscopy structural analysis with real-space, real-time high-speed atomic force microscopy imaging, we visualize the stepwise nuclease activation mechanisms of evolutionarily diverse IscB and Cas9 nucleases, including IscB.m13, type II-B PsCas9, type II-C CjCas9, and type II-D Cas9d MG34-1. We show here that the IscB family employs two distinct strategies for HNH autoinhibition, with one representing the direct evolutionary precursor of the Cas9 nuclease activation mechanism. Moreover, although the stacking-based protein-DNA interaction that stabilizes an intermediate state along the activation pathway is conserved in both IscB and Cas9 enzymes, Cas9 has acquired an additional gating element that prevents premature HNH activation immediately before catalysis. Together with previously reported structures of OgeuIscB and SpCas9, these analyses establish an atlas of the structural dynamics underlying nuclease activation across the IscB-to-Cas9 lineage, uncovering both conserved and diversified activation mechanisms and substantially advancing the mechanistic and evolutionary understanding of these RNA-guided endonucleases.
