CRISPR-Cas9 is a bacterial adaptive immune system that recently emerged as the centerpiece of a transformative genome editing technology.In this system, an intriguing allosteric communication has been suggested to control the DNA cleavage activity through the flexibility of the catalytic HNH domain. Here, accelerated Molecular Dynamics simulations are coupled to solution NMR experiments and artificial intelligence prediction tools to capture the structural and dynamic determinants of the allosteric signaling within the HNH domain. We reveal the existence of a contiguous millisecond-length dynamic pathway that enables the information transfer across the HNH domain. This pathway spans HNH from the region interfacing the RuvC nuclease and propagates up to the DNA recognition lobe in the full-length CRISPR-Cas9, such transferring the signal of DNA binding at the nuclease domains for concerted cleavages of the two DNA strands. These findings reveal the mechanism of signal transduction within the CRISPR-Cas9 HNH nuclease and pose the basis for the complete mapping of the allosteric pathway, and of its role in the DNA on-target specificity, helping engineering efforts aimed at improving the genome editing capability of CRISPR-Cas9.

Tracking the allosteric signaling in CRISPR-Cas9 by leveraging accelerated molecular dynamics, solution NMR and artificial intelligence

2019 Fall National Meeting

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