RNA Innovation Seminar: George Lisi, Brown University

“The Invisible Dance of CRISPR-Cas9 through Molecular Space and Time”
George Lisi, Ph.D.

Thomas J. & Alice M. Tisch Assistant Professor
Department of Molecular Biology, Cell Biology & Biochemistry
Brown University

In-person: BSRB, ABC seminar rooms / hybrid link

Abstract: This talk will focus on long-range signaling in CRISPR-Cas9, a cutting-edge genome editing tool. The transformative potential of Cas9 as a precision therapeutic cannot be realized without an understanding of its interdomain communication and the mitigation of deleterious off-target cleavage, which is being characterized at the atomic level in the group. A series of vignettes will highlight NMR studies of Cas9 through a “divide-and-conquer” approach using engineered protein constructs and first demonstrate that multi-timescale motions in the catalytic nuclease of Cas9 propagates chemical information that regulates cleavage of double-stranded DNA. The talk will also highlight specificity-enhancing mutations in Cas9 that rewire its regulatory mechanism and RNA interactions at the molecular level to mitigate off-target DNA cleavage. Critical energetics of the mechanism will be discussed, including the importance of metal ions and the protonation state of the catalytic histidine, studied via catalytic pocket mutations that limit conformational sampling of the Cas9 active state. Lastly, insight from canonical Cas9s will be expanded to highly stable thermophiles that are more promising for genome engineering and offer tunable high-temperature DNA editing and RNA binding. The underlying chemistry and atomic level dynamics of Cas9 will be linked to its nucleic acid interactions and biological outcomes, hopefully opening new avenues for intuitive manipulation of its function and precision therapeutic properties.