Conformational Dynamics Of Cas9 During Dna Binding.

Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA using two conserved nuclease domains, RuvC and HNH, as part of CRISPR-Cas bacterial adaptive immune systems. Together with single-guide RNAs, Cas9 is also widely utilized as a programmable genome editing tool. DNA cleavage activity is controlled directly by the conformational state of the HNH nuclease domain, but Cas9 conformational dynamics during DNA binding remain poorly characterized. Using single-molecule FRET assays, we identified a long-lived intermediate state of S. pyogenes Cas9. Upon DNA binding, the HNH nuclease reversibly transitions between RNA-bound, DNA-bound and docked conformations, before it docks irreversibly into its catalytically active conformation for DNA cleavage. Docking of HNH to its active state requires divalent cation, and HNH remains in the docked state after cleavage of a complementary target. Increasing the number of mutations in the target region distal to the protospacer adjacent motif (PAM) prevents transitions from intermediate to the docked conformation. The results provide a structural explanation for the lack of DNA cleavage activity when Cas9 binds to off-target sites.