SUBSTRATE DEPENDENT ACTIVATION OF CAS9 ENDONUCLEASE
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage.
Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites. Previously, by alanine substitutions of the non-specific DNA contacts, several Cas9 mutants have been designed to decrease the off-target activity of Cas9 while retaining the on-target activity. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown. Using single molecule FRET experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in the checkpoint state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified REC3 residues involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.