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Type of Resource (primo): dissertations
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Corporate
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Note: thesis: Thesis (Ph. D.)--Brown University, 2026
Genre (aat): theses
Abstract: The intuitive manipulation of specific amino acids or binding of exogenous ligands to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism. Furthermore, Anti-CRISPR (Acrs) are small protein inhibitors of CRISPR-Cas systems that originate from translated invading bacteriophage genetic material. By exploiting their natural ability to bind and disrupt CRISPR-Cas editing, scientists have harnessed Acr technology to leverage spatiotemporal control of editing. Recent studies have revealed how numerous and diverse the structure and function of Acrs are, with disparate Acrs having little structural or sequential similarity to each other. However, atomistic studies into the specific molecular mechanisms behind their inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition on G. stearothermophilus Cas 9(GeoCas9) by binding to GeoHNH. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface within the catalytic site and disruption of crucial electrostatic contacts responsible for modulating thermostability in GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, revealing ms motions previously absent. Uniquely, AcrIIC1 binding reduces sgRNA affinity of GeoCas9 despite previous studies in related Cas9 systems where sgRNA affinity was unaffected. Interrogation of catalytic site mutations reveal that binding of the AcrIIC1 alone is not sufficient for inhibition. Taken together, these findings provide evidence that even broad-spectrum Acrs such as AcrIIC1 exert distinct regulation of CRISPR-Cas9 systems.
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00832656")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00884652")
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