CRISPR-Cas9 is an RNA-guided endonuclease that cleavages double-stranded DNA at specific sites, and has been adapted as a powerful tool for genome manipulation. Cas9 identifies its cognate target by a series of conformational transitions coordinated between the Cas9 ribonucleoprotein and the DNA duplex. Such transitions, and consequently Cas9 target specificity, are expected to be significantly influenced by DNA shape, which refers to the collective physical properties of a duplex such as bendability and topology. However, current knowledge on the interplay between DNA shape and Cas9 target interrogation is limited. Here, we present cryo-EM structures of Cas9 bound to a cognate DNA target embedded in a relaxed 95-base-pair closed DNA double-stranded mini-circle. The Cas9/DNA segment, which was resolved to <3.0 Å, revealed the same interactions involved in PAM-binding and R-loop initiation as those observed in structures of Cas9-bound linear DNA. However, R-loop propagation was limited to 3-base-pair or less, thus significantly interfering with canonical Cas9 cleavage. Hindrance of protospacer unwinding correlated with alteration of the global DNA ring conformation beyond the Cas9-binding site, indicating that constrained and closed circular features of the DNA limited R-loop propagation. The data revealed, in details, interplay between DNA shape and Cas9 structure and function, and the insights gained could shed light on genome-editing and manipulation in complex environments where DNA topology varies.
Lee et al. (Sun,) studied this question.