Search for: All records

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. 

Abstract Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals – Mg²⁺, Ca²⁺, and Co²⁺– promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9’s metal-dependent activity. The ionic strength thereby promotes Cas9’s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms. 

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. 

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles. 

Abstract This paper presents an innovative approach for predicting the relative populations of protein conformations using AlphaFold 2, an AI-powered method that has revolutionized biology by enabling the accurate prediction of protein structures. While AlphaFold 2 has shown exceptional accuracy and speed, it is designed to predict proteins’ ground state conformations and is limited in its ability to predict conformational landscapes. Here, we demonstrate how AlphaFold 2 can directly predict the relative populations of different protein conformations by subsampling multiple sequence alignments. We tested our method against nuclear magnetic resonance experiments on two proteins with drastically different amounts of available sequence data, Abl1 kinase and the granulocyte-macrophage colony-stimulating factor, and predicted changes in their relative state populations with more than 80% accuracy. Our subsampling approach worked best when used to qualitatively predict the effects of mutations or evolution on the conformational landscape and well-populated states of proteins. It thus offers a fast and cost-effective way to predict the relative populations of protein conformations at even single-point mutation resolution, making it a useful tool for pharmacology, analysis of experimental results, and predicting evolution. 

The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs), has enabled the development of highly controllable and precise CRISPR-Cas tools. Anti-CRISPRs share very little structural or sequential resemblance to each other or to other proteins, which raises intriguing questions regarding their modes of action. Many structure–function studies have shed light on the mechanism(s) of Acrs, which can act as orthosteric or allosteric inhibitors of CRISPR–Cas machinery, as well as enzymes that irreversibly modify CRISPR–Cas components. Only recently has the breadth of diversity of Acr structures and functions come to light, and this remains a rapidly evolving field. Here, we draw attention to a plethora of Acr mechanisms, with particular focus on how their action toward Cas proteins modulates conformation, dynamic (allosteric) signaling, nucleic acid binding, and cleavage ability. 

The exquisite sensitivity of the NMR chemical shift to local environment makes it an ideal probe to assess atomic level perturbations in proteins of all sizes and structural compositions. Recent advances in solution and solid-state NMR spectroscopy of biomolecules have leveraged the chemical shift to report on short- and long-range couplings between individual amino acids to establish “networks” of residues that form the basis of allosteric pathways that transmit chemical signals through the protein matrix to induce functional responses. The simple premise that thermodynamically and functionally coupled regions of a protein (i.e. active and allosteric sites) should be reciprocally sensitive to structural or dynamic perturbations has enabled NMR spectroscopy, the premier method for molecular resolution of protein structural fluctuations, to occupy a place at the forefront of investigations into protein allostery. Here, we detail several key methods of NMR chemical shift analysis to extract mechanistic information about long-range chemical signaling in a protein, focusing on practical methodological aspects and the circumstances under which a given approach would be relevant. We also detail some of the experimental considerations that should be made when applying these methods to specific protein systems. 

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.