Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Establishing the allosteric mechanism in CRISPR-Cas9.

ABSTRACT: Allostery is a fundamental property of proteins, which regulates biochemical information transfer between spatially distant sites. Here, we report on the critical role of molecular dynamics (MD) simulations in discovering the mechanism of allosteric communication within CRISPR-Cas9, a leading genome editing machinery with enormous promises for medicine and biotechnology. MD revealed how allostery intervenes during at least three steps of the CRISPR-Cas9 function: affecting DNA recognition, mediating the cleavage and interfering with the off-target activity. An allosteric communication that activates concerted DNA cleavages was found to led through the L1/L2 loops, which connect the HNH and RuvC catalytic domains. The identification of these "allosteric transducers" inspired the development of novel variants of the Cas9 protein with improved specificity, opening a new avenue for controlling the CRISPR-Cas9 activity. Discussed studies also highlight the critical role of the recognition lobe in the conformational activation of the catalytic HNH domain. Specifically, the REC3 region was found to modulate the dynamics of HNH by sensing the formation of the RNA:DNA hybrid. The role of REC3 was revealed to be particularly relevant in the presence of DNA mismatches. Indeed, interference of REC3 with the RNA:DNA hybrid containing mismatched pairs at specific positions resulted in locking HNH in an inactive "conformational checkpoint" conformation, thereby hampering off-target cleavages. Overall, MD simulations established the fundamental mechanisms underlying the allosterism of CRISPR-Cas9, aiding engineering strategies to develop new CRISPR-Cas9 variants for improved genome editing.

SUBMITTER: Nierzwicki L

PROVIDER: S-EPMC8315640 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.

Project description:CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

| S-EPMC8741213 | biostudies-literature

Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.

Project description:BackgroundCRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

| S-EPMC7045643 | biostudies-literature

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

Project description:The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-? ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

| S-EPMC4900928 | biostudies-literature

Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage.

Project description:The CRISPR-Cas9 system from Streptococcus pyogenes has been exploited as a programmable RNA-guided DNA-targeting and DNA-editing platform. This evolutionary tool enables diverse genetic manipulations with unprecedented precision and ease. Cas9 is an allosteric enzyme, which is allosterically regulated in conformational activation, target recognition, and DNA cleavage. Here, we outline the underlying allosteric control over the Cas9 complex assembly and targeting specificity. We further review the strategies for mitigating intrinsic Cas9 off-target effects through allosteric modulations and the advances in engineering controllable Cas9 systems that are responsive to external allosteric signals. Future development of highly specific, tunable CRISPR-Cas9 systems through allosteric modulations would greatly benefit applications that require both conditional control and high precision.

| S-EPMC7308215 | biostudies-literature

Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4.

Project description:CRISPR-Cas systems act as the adaptive immune systems of bacteria and archaea, targeting and destroying invading foreign mobile genetic elements (MGEs) such as phages. MGEs have also evolved anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas systems. Recently, AcrIIC4, identified from Haemophilus parainfluenzae phage, has been reported to inhibit the endonuclease activity of Cas9 from Neisseria meningitidis (NmeCas9), but the inhibition mechanism is not clear. Here, we biochemically and structurally investigated the anti-CRISPR activity of AcrIIC4. AcrIIC4 folds into a helix bundle composed of three helices, which associates with the REC lobe of NmeCas9 and sgRNA. The REC2 domain of NmeCas9 is locked by AcrIIC4, perturbing the conformational dynamics required for the target DNA binding and cleavage. Furthermore, mutation of the key residues in the AcrIIC4-NmeCas9 and AcrIIC4-sgRNA interfaces largely abolishes the inhibitory effects of AcrIIC4. Our study offers new insights into the mechanism of AcrIIC4-mediated suppression of NmeCas9 and provides guidelines for the design of regulatory tools for Cas9-based gene editing applications.

| S-EPMC10516666 | biostudies-literature

Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6.

Project description:CRISPR-Cas systems are prokaryotic adaptive immunity against invading phages and plasmids. Phages have evolved diverse protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, to neutralize this CRISPR machinery. In response, bacteria have co-evolved Cas variants to escape phage's anti-CRISPR strategies, called anti-anti-CRISPR systems. Here we explore the anti-CRISPR allosteric inhibition and anti-anti-CRISPR rescue mechanisms between Streptococcus thermophilus Cas9 (St1Cas9) and the anti-CRISPR protein AcrIIA6 at the atomic level, by generating mutants of key residues in St1Cas9. Extensive unbiased molecular dynamics simulations show that the functional motions of St1Cas9 in the presence of AcrIIA6 differ substantially from those of St1Cas9 alone. AcrIIA6 binding triggers a shift of St1Cas9 conformational ensemble towards a less catalytically competent state; this state significantly compromises protospacer adjacent motif (PAM) recognition and nuclease activity by altering interdependently conformational dynamics and allosteric signals among nuclease domains, PAM-interacting (PI) regions, and AcrIIA6 binding motifs. Via in vitro DNA cleavage assays, we further elucidate the rescue mechanism of efficiently escaping AcrIIA6 inhibition harboring St1Cas9 triple mutations (G993K/K1008M/K1010E) in the PI domain and identify the evolutionary landscape of such mutational escape within species. Our results provide mechanistic insights into Acr proteins as natural brakes for the CRISPR-Cas systems and a promising potential for the design of allosteric Acr peptidomimetics.

| S-EPMC8632846 | biostudies-literature

Type II-C CRISPR-Cas9 Biology, Mechanism, and Application.

Project description:Genome editing technologies have been revolutionized by the discovery of prokaryotic RNA-guided defense system called CRISPR-Cas. Cas9, a single effector protein found in type II CRISPR systems, has been at the heart of this genome editing revolution. Nearly half of the Cas9s discovered so far belong to the type II-C subtype but have not been explored extensively. Type II-C CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins. Cas9s are central players in type II-C systems since they function in multiple steps of the CRISPR pathway, including adaptation and interference. Type II-C CRISPR systems are found in bacteria and archaea from very diverse environments, resulting in Cas9s with unique and potentially useful properties. Certain type II-C Cas9s possess unusually long PAMs, function in unique conditions (e.g., elevated temperature), and tend to be smaller in size. Here, we review the biology, mechanism, and applications of the type II-C CRISPR systems with particular emphasis on their Cas9s.

| S-EPMC5815938 | biostudies-literature

Peptide dendrimers transfecting CRISPR/Cas9 plasmid DNA: optimization and mechanism.

Project description:Gene editing by CRISPR/Cas9 offers great therapeutic opportunities but requires delivering large plasmid DNA (pDNA) into cells, a task for which transfection reagents are better suited than viral vectors. Here we performed a structure-activity relationship study of Z22, a d-enantiomeric, arginine containing, lipidated peptide dendrimer developed for pDNA transfection of a CRISPR/Cas9 plasmid co-expressing GFP. While all dendrimer analogs tested bound pDNA strongly and internalized their cargo into cells, d-chirality proved essential for transfection by avoiding proteolysis of the dendrimer structure required for endosome escape and possibly crossing of the nuclear envelope. Furthermore, a cysteine residue at the core of Z22 proved non-essential and was removed to yield the more active analog Z34. This dendrimer shows >83% GFP transfection efficiency in HEK cells with no detrimental effect on cell viability and promotes functional CRISPR/Cas9 mediated gene editing. It is accessible by solid-phase peptide synthesis and therefore attractive for further development.

| S-EPMC11352961 | biostudies-literature

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.

Project description:CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.

| S-EPMC7497131 | biostudies-literature