Biology subjects

Palermo, G.

Publications and source records attributed to Palermo, G..

18 recordsLinked to original sources

bioRxiv2026

Edge-Aware Graph Attention Networks for Interpreting Biophysical Mechanisms from Molecular Dynamics Simulations

Graph attention networks (GATs) are emerging as powerful aritficial intelligence (AI) tools for learning biomolecular dynamics, yet extracting mechanistic insight from learned attention remains challenging. Here, we present an interpretable AI approach that combines molecular dynamics prediction with mechanistic interpretation through attention-derived communication networks. We develop three edge-aware GAT models - Edge-Conditioned, Edge-Injected, and Edge-Gated - that differ in how edge information is incorporated during message passing. Relative to a standard GAT, our edge-aware models improve coordinate prediction while recovering complementary aspects of residue communication. Application to Chignolin and HIV-1 protease demonstrates their ability to characterize communication networks underlying protein folding, allostery, and mutation-induced functional remodeling. Analysis of the communication networks using graph-based descriptors of communication throughput, intensity, and relay revealed that the Edge-Conditioned model preferentially emphasizes communication hubs characterized by high communication throughput and intensity, the Edge-Injected model preferentially highlights high-intensity communication within functionally important regions, and the Edge-Gated model most clearly resolves long-range communication relay. Overall, this approach provides an interpretable AI strategy for uncovering the communication mechanisms that underlie folding, allostery, and long-range signal propagation in molecular machines, while also supporting AI-guided modulation of biomolecular function and rational protein engineering.

biophysics↗

bioRxiv2026

Transposon end recognition and pairing by I-F3 CRISPR-associated transposase

To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Tn7-like transposons typically contain asymmetric left- and right-end sequences that flank and define DNA cargo. However, how the transposase recognizes these different sequences and assembles them into a paired end complex for cut-and-paste transposition remains unknown. Here we present the cryo-EM structure of type I-F3 (VchCAST) CAST transposase TnsB in complex with transposon DNA ends and host factor IHF, along with biochemistry, molecular dynamics, and in vivo analyses. Our structure reveals the stoichiometry and architecture of the assembly, as well as the DNA distortions required to accommodate transposon end asymmetry. Molecular dynamics suggests that these distortions are required for the coordinated assembly of the complex. Physical pairing of asymmetric left and right ends results in a novel protein-protein interface that is required for transposition efficiency. Our findings explain how transposases regulate the pairing of transposon end sequences for high-fidelity recognition, reveal a model of transposon end synapsis, and suggest future avenues to engineer DNA cargo for genome-editing applications.

biochemistry↗

bioRxiv2026

Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on GeoCas9 from integrated structural biophysics

Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (GeoCas9) via its HNH nuclease domain. An X-ray crystal structure of the GeoHNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of GeoCas9. AcrIIC1 binding also rewires the intrinsic dynamics of the GeoHNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of GeoCas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on GeoCas9.

biophysics↗

bioRxiv2026

Massive-scale single-nucleus multi-omics identifies novel rare noncoding drivers of Parkinson's disease

Most genetic variants contributing to complex diseases reside in the noncoding genome. While common variants uncovered by genome-wide association studies often fail to explain much of the observed heritability of these diseases, rare variants often have higher effect sizes and cumulatively explain a larger portion of heritability. However, rare variants, particularly rare noncoding variants, have remained under-characterized largely due to the difficulties of accurately predicting variant functionality at scale, given that each individual carries an average of [~]10,000 rare variants. Here, we generated multi-omic data from >3.3 million nuclei sampled from five brain regions across a cohort of 80 individuals with Parkinsons disease (PD) and 21 neurologically normal control individuals with matched 30x whole-genome sequencing. We use this data to identify cell type-specific features of PD, map cell type-specific chromatin accessibility and expression quantitative trait loci, and train machine learning models to predict the effect of variants on gene regulation. We identify rare noncoding variants statistically associated with sporadic PD and extend our approaches to predict drivers of familial PD of unknown genetic origin. Our results underscore the significance of rare noncoding variants in complex diseases and provide a roadmap for applying similar approaches in other disease systems.

genetics↗

bioRxiv2025

Tuning Histone Site Selectivity in the Nucleosome with Organometallic Agents

Platinum anticancer drugs tend to target DNA whereas certain ruthenium and osmium organometallic compounds, including those with known anticancer activity, preferentially bind histone proteins in chromatin. We earlier found that Ru/Os arene 2-pyridinecarbothioamide antitumor agents display unique or partially overlapping profiles of histone protein binding in the nucleosome compared to Ru arene phosphaadamantane (RAPTA) antimetastasis drugs, but the basis for this difference is unclear. Here we structurally characterized the nucleosome binding effects of arene ligand substitutions and carried out a multiscale simulation analysis, which reveals that the interplay between metal cation and non-leaving ligand identity dictates adduct stability and whether complexes target electronegative surface patches, internal crevices, or both. We show that the nucleosome superhelical crevice acts as a small molecule selectivity filter and that multi-site binding profiles can be expanded or reduced through defined ligand substitutions, which modulate dynamic and steric attributes. Our findings suggest new avenues for rationally developing Ru/Os organometallics that could help expand the scope of chromatin-targeting therapeutics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/688318v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@188bc1dorg.highwire.dtl.DTLVardef@1f619d5org.highwire.dtl.DTLVardef@1a0ebaorg.highwire.dtl.DTLVardef@bd0abb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/673821v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1011b93org.highwire.dtl.DTLVardef@1b96a0eorg.highwire.dtl.DTLVardef@1e1c42corg.highwire.dtl.DTLVardef@11628be_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

A Cryptic Binding Pocket Regulates the Metal-Dependent Activity of Cas9

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 Cas9s metal-dependent activity. The ionic strength thereby promotes Cas9s 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.

biophysics↗

bioRxiv2025

Graph Attention Neural Networks Reveal TnsC Filament Assembly in a CRISPR-Associated Transposon

CRISPR-associated transposons (CAST) enable programmable, RNA-guided DNA integration, marking a transformative advancement in genome engineering. A central player in the type V-K CAST system is the AAA+ ATPase TnsC, which assembles into helical filaments on double-stranded DNA (dsDNA) to orchestrate target site recognition and transposition. Despite its essential role, the molecular mechanisms underlying TnsC filament nucleation and elongation remain poorly understood. Here, multiple-microsecond and free energy simulations are combined with deep learning-based Graph Attention Network (GAT) models to elucidate the mechanistic principles of TnsC filament formation and growth. Our findings reveal that ATP binding promotes TnsC nucleation by inducing DNA remodelling and stabilizing key protein-DNA interactions, particularly through conserved residues in the initiator-specific motif (ISM). Furthermore, GNN-based attention analyses identify a directional bias in filament elongation in the 5'[->]3' direction and uncover a dynamic compensation mechanism between incoming and bound monomers that facilitate directional growth along dsDNA. By leveraging deep learning-based graph representations, our GAT model provides interpretable mechanistic insights from complex molecular simulations and is readily adaptable to a wide range of biological systems. Altogether, these findings establish a mechanistic framework for TnsC filament dynamics and directional elongation, advancing the rational design of CAST systems with enhanced precision and efficiency.

biophysics↗

bioRxiv2025

CRISPR-Cas12a REC2 - NUC interactions drive target-strand cleavage and constrain trans cleavage.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis, after which it remains catalytically active and non-specifically cleaves ssDNA in trans. Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied NUC loop, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/644851v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@18c702aorg.highwire.dtl.DTLVardef@11816d1org.highwire.dtl.DTLVardef@822316org.highwire.dtl.DTLVardef@1cda6bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Mechanism of expanded DNA recognition in xCas9

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.

biophysics↗

bioRxiv2024

Unveiling Cas8 Dynamics and Regulation within a transposon-encoded Cascade-TniQ Complex

The Vibrio cholerae Cascade-TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of "knocking in" genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open-to-close conformational transition at key steps of the complexs function. We show that when Cascade-TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction alleviates unfavorable contacts and synchronizes Cas8s shift with neighboring subunits, lowering the barrier for the transition to the open state, a critical requirement for DNA binding. As DNA fully pairs with RNA, the open state becomes increasingly accessible, favoring interactions with DNA and aiding the formation of an R-loop. These outcomes provide the first dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade-TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms.

biophysics↗

bioRxiv2023

High-Fidelity, Hyper-Accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9

The Cas9-HF1, HypaCas9, and evoCas9 variants of the Cas9 endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in the Rec3 domain mediate specificity in these variants are poorly understood. Here, solution NMR 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 peculiar dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic HNH domain. The variants remodel the communication sourcing from the Rec3 -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.

biophysics↗

bioRxiv2023

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTSO_LICertain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches. C_LIO_LIUnderstanding Cas13as allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants. C_LIO_LIThese Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/550755v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a3fd63org.highwire.dtl.DTLVardef@1509a82org.highwire.dtl.DTLVardef@cfcd7aorg.highwire.dtl.DTLVardef@bb2f0f_HPS_FORMAT_FIGEXP M_FIG New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a. C_FIG

molecular biology↗

bioRxiv2023

RNA-mediated Allosteric Activation in CRISPR-Cas13a

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two Higher Eukaryotes and Prokaryotes Nucleotide (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.

biophysics↗

bioRxiv2022

An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a

CRISPR-Cas12a is a powerful RNA-guided genome-editing system, also emerging as a robust diagnostic tool that cleaves double-stranded DNA using only the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an -helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:TS hybrid toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. Understanding of this cardinal process in the functioning of Cas12a will enrich fundamental knowledge and facilitate further engineering strategies for genome-editing.

biophysics↗

bioRxiv2021

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

CRISPR-Cas9 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, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the 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 HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the systems specificity.

biophysics↗

bioRxiv2019

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

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 timescale 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.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC=\"FIGDIR/small/660613v4_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (64K):\norg.highwire.dtl.DTLVardef@13429c5org.highwire.dtl.DTLVardef@1a2493borg.highwire.dtl.DTLVardef@175e120org.highwire.dtl.DTLVardef@f1852_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2018

Molecular mechanism of off-target effects in CRISPR-Cas9

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations - using Gaussian accelerated MD (GaMD) - are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a \"lock\" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to \"lock\" the HNH domain, thereby identifying the ability to \"lock\" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to \"trap\" somehow the HNH domain in an inactive \"conformational checkpoint\" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the \"locking\" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

biophysics↗