The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as an efficient genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants can be programmed with single guide RNAs to site-specifically recognize and specifically cleave double stranded DNA sequence, thus enabling a wide range of applications. In spite of a broad exploitation for modifying the genome, the structural and mechanistic details underlying its function are incompletely understood. Indeed, while X-ray crystallography and electron microscopy portrayed Cas9 in different conformational states, the mechanism of conformational transition activating the catalytic machinery is unclear. Here, we employ a variety of computational methods – including long-time scale molecular simulations, biasing methods and accelerated MD – to investigate the mechanism of conformational activation of the Cas9 endonuclease along its activation trajectory, from the apo protein via the binary Cas9-guide RNA intermediate, up to the target DNA-bound complexes. By characterizing the conformational dynamics of Cas9 over the micro–<i>to</i>–millisecond time scale, we reveal that a remarkable conformational flexibility of the protein framework is essential for nucleic acid association and catalysis. We show how the “closure” of the protein, which accompanies the nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains that collectively initiate the prominent conformational changes necessary for the nucleic acid association. We further disclose that a tight interplay between Cas9 and the nucleic acids orchestrates the protein activation toward DNA cleavage, revealing how the catalysis depends by the relocation of the nucleic acids. Overall, our outcomes address the lack of mechanistic information on CRISPR-Cas9 and constitute a fundamental reference for future experimental work aimed at understanding this unique genome editing machinery.

Unraveling the mechanistic basis of the CRISPR-Cas9 system via molecular simulations

253rd National Meeting (2017)

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