Project Summary The PI Batista from Yale and co-investigators (Lisi, Brown University, and Palermo, UC Riverside) will investigate allosteric pathways in the CRISPR-Cas9 system – composed of the multi-domain endonuclease Cas9 in complex with RNA and DNA. The system allows for studies of long-range signaling critical for allosteric mechanisms that achieve enhanced selectively and tunability of the protein/nucleic acid complex response. CRISPR-Cas9 is an innovative therapeutic tool with widely demonstrated capabilities for genome editing. An outstanding challenge of great research interest is to develop a detailed understanding of allosteric signals in CRISPR-Cas9 responsible for the DNA editing capability. Such understanding would have profound implications for bioengineering and precision medicine, as well as for establishing modern paradigms of allosteric regulation in protein/nucleic acid machines. A substantial hurdle in investigating the mechanisms of large protein/nucleic acid complexes is the inherent difficulty of adapting experimental and computational methodologies to capture the intrinsic flexibility of these structures essential for functionality. We propose to implement a synergistic approach of solution NMR and molecular dynamics (MD) in combination with established and novel methods for analysis of allosteric networks to elucidate the structural and dynamic determinants of allosteric signaling in CRISPR-Cas9. We have recently identified a pathway of dynamic communication connecting multiple domains of Cas9 through millisecond timescale motion that spans its critical nucleases, consistent with a regulatory signal proposed through experimental characterization. Thus, the following hypotheses guide our specific aims: (i) A well-defined allosteric pathway controls the CRISPR-Cas9 functionality; (ii) The allosteric interplay between spatially distant protein domains activates the DNA nuclease function; (iii) Modulation of the allosteric motions through the mutation of critical residues achieves altered specificity; and (iv) Dynamically-driven signaling is an intrinsic property of protein-nucleic acid macromolecular complexes. Our specific aims are: Aim 1: Characterize the allosteric control of the HNH nuclease; Aim 2: Determine the allosteric pathway from HNH to RuvC and the allosteric role of the PAM recognition sequence; and Aim 3: Characterize the effect of mutations on the allosteric pathway. The research program involves multiple cycles of an iterative approach where, in each cycle, allosteric pathways are explored through the analysis of differential motions probed by liquid-NMR relaxation methods and computation (MD and network analysis), obtaining valuable information on key amino acid residues and specific interactions responsible for transmitting structural or dynamical changes spanning the allosteric and active sites. The resulting insight provides guidelines for the next round of studies of mutants and modulators in a joint experimental and theoretical effort to elucidate the CRISPR-Cas9 allosteric mechanisms.

Project Narrative State-of-the-art NMR techniques, site-directed mutagenesis and computational studies based on microsecond molecular dynamics/network analysis, and simulations of NMR spectra will explore allosteric pathways in CRISPR- Cas9, an innovative therapeutic tool with widely demonstrated capabilities for genome editing.