Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology has linked numerous pathologies to dysregulation of these processes. Although modification of the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR-Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations, however, to fully realize these applications, novel strategies to overcome its off-target effects and poor temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif (PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9 nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer little mechanistic information, as the target DNA and catalytic nucleases are never observed in an activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision medicine by establishing novel paradigms to control and enhance the spatial and temporal function of Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric network that controls Cas9 function. To investigate the reach of this allosteric network and the role of molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and computational study to map the longrange allosteric pathway of Cas9. We will (1) characterize the molecular determinants of protein motions in the HNH nuclease, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in Cas9 signal transduction and (3) characterize the interaction of the PAM sequence with its binding domain to evaluate its role as an allosteric activator. Specifically, this multidisciplinary approach of NMR spin relaxation experiments and molecular dynamics, network theory, and Eigenvector Centrality simulations will probe differential protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information to affect biological response. These studies will use both full-length Cas9 and novel engineered constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic findings of this work will be correlated to function with biochemical and cellular assays to provide a detailed understanding of the Cas9 allosteric mechanism.

CRISPR-Cas9 has potential to modify disease-causing genes, but is prone to off-target alterations due to poor temporal control its expression. It is therefore desirable to develop an allosterically-controlled Cas9 that elicits no function unless activated, circumventing this limitation. Cas9 is reliant on conformational dynamics for allosteric function, but typical solution methods for characterizing motional ensembles, namely NMR, are insufficient as standalone techniques to fully characterize an enzyme of this size. Pairing experiments with in silico methods can elevate the level of insight from NMR alone by more precisely modeling NMR data and generating dynamic structural networks that illuminate regions of allosteric crosstalk that may become functional handles for enhanced control over genome editing.