Structures of Cas9-Bound Double-Stranded DNA Mini-Circle Reveal Impacts of DNA Shape on Cas9 Target Interrogation

CRISPR-Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA at specific sites and has been adapted as a powerful tool for genome manipulation. Cas9 recognizes its target through multiple conformational transitions coordinated between the Cas9 ribonucleoprotein and the DNA duplex. Such transitions, and consequently Cas9 targeting specificity, are expected to be significantly influenced by the collective duplex physical properties referred to as DNA shape. To advance our currently limited understanding of the interplay between DNA shape and Cas9 target interrogation, we solved two cryo-EM structures of SpyCas9 bound to a cognate target embedded in a relaxed 95-base-pair DNA double-stranded minicircle. The Cas9-bound DNA segment engages in similar interactions involved in PAM-binding and R-loop initiation as those observed in Cas9-bound linear DNA. However, R-loop is limited to less than three base-pairs, thus interfering with Cas9 cleavage. The minicircle DNA, which is fully resolved, retains its global shape. As Cas9 locally unwinds the protospacer, the closed-ring topology constrains the movement of the paired PAM-distal DNA duplex, thus interfering with R-loop propagation. These data provide detailed insight into the interplay between DNA shape and Cas9 structure and function, and may shed light on genome-editing and manipulation in environments with varied DNA topologies.