PT - JOURNAL ARTICLE AU - Pacesa, Martin AU - Jinek, Martin TI - Mechanism of R-loop formation and conformational activation of Cas9 AID - 10.1101/2021.09.16.460614 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.09.16.460614 4099 - http://biorxiv.org/content/early/2021/09/16/2021.09.16.460614.short 4100 - http://biorxiv.org/content/early/2021/09/16/2021.09.16.460614.full AB - Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.Competing Interest StatementThe authors have declared no competing interest.