Structure and dynamics of off-target effects in CRISPR-Cas9

The occurrence of off-target mutations in CRISPR-Cas9 can limit the applicability of this technology in vivo. At the molecular level, off-target effects are the unselective cleavage of DNA sequences that do not fully match the guide RNA in Cas9. Using enhanced simulation methods, we probed the effect of DNA base pair mismatches in the dynamics of CRISPR-Cas9. We found that, depending on their position and nature, base pair mismatches can induce an opening of the RNA:DNA hybrid, which results in newly formed interactions with the catalytic HNH domain. These interactions can “lock” the HNH domain in an inactive state, exerting a conformational control. These findings were corroborated by several X-ray structures of Cas9 bound to off-target substrates. Based on this success, we used molecular simulations to predict the effect of mismatches at positions that were not captured in the crystallographic structures, providing a more complete overview of the structural and dynamic effects of DNA mismatches in Cas9. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the rational design of guide RNAs and off-target prediction algorithms.