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Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency. 

Metal ions play a critical role in various chemical, biological, and environmental processes. This review reports on emerging chemical mechanisms in the catalysis of DNA and RNA. We provide an overview of the metal-dependent mechanisms of DNA cleavage in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems that are transforming life sciences through genome editing technologies, and showcase intriguing metal-dependent mechanisms of RNA cleavages. We show that newly discovered CRISPR-Cas complexes operate as protein-assisted ribozymes, highlighting RNA's versatility and the enhancement of CRISPR-Cas functions through strategic metal ion use. We demonstrate the power of computer simulations in observing chemical processes as they unfold and in advancing structural biology through innovative approaches for refining cryo-electron microscopy maps. Understanding metal ion involvement in nucleic acid catalysis is crucial for advancing genome editing, aiding therapeutic interventions for genetic disorders, and improving the editing tools’ specificity and efficiency. 

The intuitive manipulation of specific amino acids 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 ofGeoCas9, a thermophilic Cas9 fromGeobacillus stearothermophilus. Our results provide new atomic details about theGeoRec subdomains (GeoRec1,GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughoutGeoRec, and NMR studies of the interaction betweenGeoRec 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 ofGeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilicStreptococcus pyogenesCas9. A K267E/R332A double mutant did also did not enhanceGeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe ofGeoCas9 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 ofGeoCas9, setting the stage for future studies ofGeoCas9 variants and their effect on its allosteric mechanism. 

Abstract The intuitive manipulation of specific amino acids 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. 

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.