The RNA-programmable CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a genome editing technology with transformative impact in life sciences. In recent years, extensive biophysical studies delivered fundamental information on the structural and conformational requirements for nucleic acids binding and cleavage. However, the mechanism underlying the formation of a catalytically active system, which is prone for catalysis, remains incompletely understood. Here, molecular dynamics (MD) – performed over ~16 μs of continuous simulations – reveal the activation process, showing that the approach of the catalytic domain HNH to the DNA cleavage site is accompanied by the structural remodeling of the α-helical lobe. The remarkable conformational changes and the collective conformational dynamics of the α-helical components are observed on an atomic scale, providing information on how the recognition regions Rec1-3 enable “sensing” nucleic acids and “regulating” the HNH conformational transition, as well as “locking” HNH at the cleavage site, contributing in its catalytic competence. This work deciphers the mechanistic role of the α-helical components in the CRISPR-Cas9 activation process. Considering the key role of the α-helical lobe in the specificity of Cas9, this study poses the basis for fully understanding how the α-helical components control the cleavage of off-target sequences. Overall, this study lays the foundation for future mechanistic studies and novel engineering efforts toward improved genome editing applications.

Formation of an activated CRISPR-Cas9 by “sensing”, “regulating” and “locking” the catalytic HNH domain

256th National Meeting (2018)

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