Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology techniques have linked numerous pathologies to dysregulation of genes. However, faithful modification of the genome in studies of pathogenic mutations and associated mechanisms have been difficult, rarely leading to effective treatments. The advent of CRISPR-Cas9 (Cas9) as an inexpensive tool for genome editing has created new possibilities for therapeutic gene targeting. Cas9 has potential to permanently correct disease-linked genetic mutations and deconvolute the underlying biology, but to fully harness Cas9 function, the structural underpinnings of its catalytic mechanism must be elucidated. Cas9 utilizes guide RNA to cleave complementary double-stranded DNA upon binding of a Protospacer Adjacent Motif (PAM), a key genomic recognition sequence. Two nuclease domains within Cas9, HNH and RuvC, cleave the DNA strands and early biophysical studies suggest conformational changes within the nucleases upon RNA and DNA binding are functionally relevant. However, X-ray crystal structures offer little information about the activated state of Cas9, as the catalytic HNH domain and its target DNA strand are shown 20 Å apart. Interestingly, this HNH conformational shift is closely correlated to the enhancement of RuvC nuclease activity, suggesting these spatially separated domains are functionally coupled by an allosteric mechanism. The molecular motions associated with interdomain signaling have not been clarified, and the PI hypothesizes that allosteric communication, and ultimately nuclease function, in Cas9 is driven by structural dynamics on multiple timescales. The hypothesis will be investigated with a synergistic solution nuclear magnetic resonance (NMR) and computational approach to assess the contribution of conformational dynamics to long-range allosteric signaling in Cas9. A detailed understanding of this mechanism will facilitate greater structural control of Cas9 and help to circumvent current limitations in its genome editing power, most notably errors due to off-target nucleotide mismatches and poor temporal control of Cas9 activity. Our initial studies of Cas9 conformational dynamics will use novel constructs that facilitate interrogation of specific domains within the 160 kDa molecular machine that is nearly inaccessible with biomolecular NMR alone. Our preliminary data is the first evidence of micro-millisecond protein motions in the HNH nuclease, consistent with dynamically-regulated endonuclease activity. Foundational work completed during the funding period will implement novel computational methodologies that enhance our experimental insight to provide an atomistic glimpse of the Cas9 allosteric pathway. These results can be leveraged in subsequent expansions of the work toward targeting genes implicated in disease.
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 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 pushing their limits as standalone techniques for enzymes of this size. Pairing experiments with in silico methods can elevate the level of insight from NMR alone by more precisely treating NMR data and generating dynamic structural networks that illuminate regions of allosteric crosstalk that may become functional handles for enhanced control over genome editing.