CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Abstract

<p id="d949277e285">The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9) system recently emerged as a transformative genome editing technology that is innovating life science, with cutting-edge impact in biomedicine, pharmaceutics, and agriculture. Nevertheless, the molecular mechanism underlying CRISPR-Cas9 function is still incompletely understood. Here, enhanced molecular dynamics (MD) simulations, probing displacements over long timescales, capture at atomic level specific features that are difficult to reach via conventional MD simulations and via the currently available experimental techniques, clarifying the molecular mechanism of CRISPR-Cas9, with understanding of its activation process. The insights obtained from our molecular simulations provide key reference points for future experimental studies of CRISPR-Cas9 and its applications as a genome editing tool. </p><p class="first" id="d949277e288">CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) <i>Nature</i>, <i>527</i>, 110–113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9. </p>