Allosteric Motions of the CRISPR–Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

Abstract

CRISPR–Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR–Cas9. These findings reveal a potential route of signal transduction within the CRISPR–Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR–Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.

Establishing the allosteric mechanism in CRISPR‐Cas9

“…[56][57][58] The residues involved in this allosteric pathway were further shown by NMR experiments to give rise to slow millisecond-length relaxations, thereby supporting the theoretical finding of a long timescale dynamical route of communication between REC and RuvC. 54 Notably, the theoretical pathway also encompassed two critical lysine residues (K855, K810), which anchor HNH at the DNA strands and whose alanine mutation is key for the enhanced specificity Cas9 variants. 38 This further supports the hypothesis that protein allostery intervenes with the specificity of Cas9, supporting the early suggestion that the allosterism can be modulated to improve the CRISPR-Cas9 function.…”

“…15 To ultimately track the allosteric routes transferring the information from REC to HNH and ultimately RuvC, GaMD was combined with graph theory. 54 At first, a series of 400 ns GaMD simulations were successfully used to recover the transitions of HNH observed over μs using Anton2 (Figure 4a). Then, the simulated ensemble was subjected to the analysis of the allosteric signaling through graph theory.…”

Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9

“…The method has been shown to enhance a broad sampling of the conformational space in large biomolecular systems McCammon, 2016, 2018;Wang and Chan, 2017;Liao and Wang, 2018;Sibener et al, 2018), including CRISPR-Cas9 as apo form and in complex with nucleic acids (Palermo et al, 2017;Palermo, 2019b), or bound to off-target DNAs (Ricci et al, 2019). Recently, GaMD has shown to sample long time scale motions in agreement with NMR relaxation experiments, showing that the method can efficiently capture the dynamics of large protein/nucleic acid complexes (East et al, 2020). A set of model systems have been built; introducing couples of base pair mismatches "mm" within the hybrid complex at positions 10 to 17 (i.e., mm@10-11, @12-13, @14-15, and @16-17, Figure 1A, bottom panel).…”

Backbone-independent NMR resonance assignments of methyl probes in large proteins

“…6 and 7 ) and used conventional methods to obtain reference backbone and methyl assignments for validating the MAUS results ( Supplementary Results ). Concurrently with the development of the present work, the backbone and side-chain assignments for the HNH domain were obtained using a conventional backbone-based approach and released in the Biological Magnetic Resonance Data Bank (BMRB, entry 27949) 20 , 21 . The MAUS-derived methyl assignments were in full agreement with the published results, which further highlights the practical utility of our method in saving machine time and manual effort spent.…”