CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, enhanced molecular dynamics (MD) simulations are used to decipher the mechanism of off-target binding at the molecular level. The simulations reveal that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the catalytic HNH domain. These conserved interactions constitute a “lock”, effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the non-specific cleavage of DNA sequences. Consistent with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes to clarifying the onset of off-target effects in CRISPR-Cas9 and provides fundamental information for designing novel and more specific Cas9 variants. Such variants with enhanced specificity could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Deciphering off-target effects in CRISPR-Cas9 through molecular dynamics

ACS Fall 2021

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