A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Abstract

The Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. 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.

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

“…Indeed, by sampling multiple configurations, the interconnecting regions reorient the HNH domain in the active state, driving its catalytic activation. Moreover, the active states of the HNH domain are characterized by the presence of the nt-DNA strand within the RuvC cleft, indicating that the activation of the HNH toward cleavage of the t-DNA domain requires the presence of the nt-DNA within the RuvC cleft (12,13). In the inactive states (both Cas9:RNA and Cas9:DNA), the HNH domain samples a distinct set of configurations, in which the catalytic H840 points in the opposite direction with respect to the t-DNA and which are separated from the active states (Fig.…”

“…However, due to the short timescales accessible by conventional MD, this mechanistic study could not fully characterize the long-timescale conformational transitions arising from the interplay between protein and nucleic acids (12,14).…”

“…Moreover, information about the conformational activation of the HNH domain is elusive. Förster resonance energy transfer (FRET) experiments have shown that the conformational activation of the HNH domain controls DNA cleavage (11,12), but it is unclear how the protein structural changes in concert with DNA binding would allow its conformational activation toward catalysis. By using molecular…”

A Broad-Spectrum Inhibitor of CRISPR-Cas9

“…Further analysis of AcrIIC1 binding to NmeCas9 by isothermal titration calorimetry (ITC) demonstrated an equilibrium binding affinity of AcrIIC1 6.3 ± 3.4 nM with a stoichiometry of one AcrIIC1 for each Cas9 (Figure S3D). The HNH nuclease domain is highly conserved across all Cas9 proteins (Figure S3E) and controls cleavage of both strands of the target DNA (Dagdas et al, 2017; Sternberg et al, 2015). Although the Cas9 HNH nuclease domain is directly responsible for cleavage of the target strand of the DNA (Gasiunas et al, 2012; Jinek et al, 2012), conformational activation of the HNH domain is a prerequisite for activating cleavage of the non-target strand by the RuvC nuclease domain (Sternberg et al, 2015).…”

“…The best understood of these checkpoints is mediated by the HNH domain, which undergoes a large rotation and translation to cleave the target DNA only when sufficient complementarity to the guide RNA is sensed (Dagdas et al, 2017; Sternberg et al, 2015). The structure presented here suggests that AcrIIC1 exploits one checkpoint in this process to ensure inhibited cleavage of both the target and non-target DNA strands.…”