Biology subjects

Jung, K.

Publications and source records attributed to Jung, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Depletion of m6A-RNA in Escherichia coli reduces the infectious potential of T5 bacteriophage

N6-methyladenosine (m6A) is the most abundant internal modification of mRNA in eukaryotes that plays, among other mechanisms, an essential role in virus replication. However, the understanding of m6A RNA modification in prokaryotes, especially in relation to phage replication, is limited. To address this knowledge gap, we investigated the effects of m6A RNA modification on phage replication in two model organisms: Vibrio campbellii BAA-1116 (previously V. harveyi BB120) and Escherichia coli MG1655. An m6A-RNA depleted V. campbellii mutant ({Delta}rlmF{Delta}rlmJ) did not differ from the wild type in the induction of lysogenic phages or in susceptibility to the lytic Virtus phage. In contrast, the infection potential of the T5 phage, but not that of other T phages or the lambda phage, was reduced in an m6A-RNA depleted E. coli mutant ({Delta}rlmF{Delta}rlmJ) compared to the wild type. This was shown by a lower efficiency of plaquing and a higher percentage of surviving cells. There were no differences in T5 phage adsorption rate, but the mutant exhibited a 5 min delay in the rise period during the one-step growth curve. This is the first report demonstrating that E. coli cells with lower m6A RNA levels have a higher chance of surviving T5 phage infection. ImportanceThe importance of RNA modifications has been thoroughly studied in the context of eukaryotic viral infections. However, their role in bacterial hosts during phage infections is largely unexplored. Our research delves into this gap by investigating the effect of host m6A-RNA modifications during phage infection. We found that an E. coli mutant depleted of m6A-RNA is more resistant to T5 infection than the wild type. This finding emphasizes the need to further investigate how RNA modifications affect the fine-tuned regulation of individual bacterial survival in the presence of phages to ensure population survival.

microbiology↗

bioRxiv2024

The chemoreceptor controlling the Wsp-like transduction pathway in Halomonas titanicae KHS3 binds purine derivatives

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of the pathway results in colony morphology alterations and increased ability to form biofilm. Such characteristics resemble the behaviour of the Wsp pathway of Pseudomonas. In this work we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. Thermal shift analyses performed with the Htc10 ligand-binding domain led to the identification of purine derivatives as ligands. This ligand-binding domain was crystallized in the presence of guanine or hypoxanthine and its structure was solved by X-ray protein crystallography. The sensor domain adopts a double-cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocket allowed the identification of the involved residues. These residues were validated by site directed mutagenesis and thermal shift or isothermal calorimetry analyses of the protein variants. The dissociation constants for guanine or hypoxanthine of the intact domain were in the low micromolar range. To our knowledge, this is the first description of binding specificity for a chemoreceptor that controls the activity of an associated diguanylate cyclase, and opens the way for dynamic studies of the signalling behaviour of this kind of sensory complex. A comparison between Htc10 and the functionally equivalent WspA receptor from Pseudomonas revealed no significant sequence similarities. In contrast, highly conserved Htc10-like receptors were found in distant bacteria carrying HtChe2-like clusters.

microbiology↗

bioRxiv2024

Frontal engagement in perceptual integration under low subjective visibility

We investigated how spatiotemporal neural dynamics underlying perceptual integration changed with the degree of conscious access to a set of backward-masked pacman-shaped inducers that generated the percept of an illusory triangle. We kept the stimulus parameters at a fixed near-threshold level throughout the experiment and recorded electroencephalography from participants who reported the orientation and subjective visibility of the illusory triangle on each trial. Our multivariate pattern analysis revealed that posterior and central regions initially used dynamic neural code and later switched to stable neural code. The transition from dynamic to stable neural code in posterior region occurred increasingly later and eventually disappeared with decreasing conscious access. Anterior region primarily used stable neural code which waned with decreasing conscious access, but increased at below-median visibility and remained even when stimulus awareness was minimal. These results demonstrate differential spatiotemporal neural dynamics underlying perceptual integration depending on conscious access and emphasize a unique role of anterior region in processing integrated shape information especially under low subjective visibility.

neuroscience↗

bioRxiv2024

Simulated brain networks reflecting progression of Parkinson's disease

Neurodegenerative progression of Parkinsons disease affects brain structure and function and, concomitantly, alters topological properties of brain networks. The network alteration accompanied with motor impairment and duration of the disease is not yet clearly demonstrated in the disease progression. In this study, we aim at resolving this problem with a modeling approach based on large-scale brain networks from cross-sectional MRI data. Optimizing whole-brain simulation models allows us to discover brain networks showing unexplored relationships with clinical variables. We observe that simulated brain networks exhibit significant differences between healthy controls (n=51) and patients with Parkinsons disease (n=60) and strongly correlate with disease severity and disease duration of the patients. Moreover, the modeling results outperform the empirical brain networks in these clinical measures. Consequently, this study demonstrates that utilizing simulated brain networks provides an enhanced view on network alterations in the progression of motor impairment and potential biomarkers for clinical indices.

neuroscience↗

bioRxiv2023

Commentary on Pang et al. (2023) Nature

Pang et al. (2023) observe that the geometric eigenmodes, derived from the shape of the cortical surface, are better at reconstructing patterns of both spontaneous and stimulus-evoked activity, when contrasted with three alternative connectome-based models including structural connectome derived eigenmodes. Based on this observation they propose that geometric eigenmodes offer a good model for explaining brain function, noting that "wave dynamics offer a more accurate and parsimonious mechanistic account of macroscale, spontaneous cortical dynamics captured by fMRI". They then question the prevailing view that brain activity is "localized to focal, spatially isolated clusters" and it is driven by "intricate patterns of anatomical connections". While the observation that geometric properties fit brain activity well is intriguing, we argue that accepting geometric eigenmodes as a model for brain function risks the logical fallacy of "affirming the consequent". A representation that effectively describes the underlying geometry is inherently adept at fitting patterns within that geometric space; it does not necessarily shed light on mechanisms of the brains functional attributes. To this end, we provide two lines of empirical results: (a) Basic parcel-based representations, which capture localized structures, can reconstruct activity patterns as well. (b) Geometric eigenmodes demonstrate a high flexibility when fit to a range of manipulated patterns, which evokes the danger of overfitting. Based on those results, theoretical considerations, and previous data we argue that more consideration is needed regarding "parsimony, robustness and generality of geometric eigenmodes as a basis set for brain function". While we recognize the potential role of the brains geometry in influencing its dynamics, assertions regarding its efficacy should be weighed against the performance of simpler models, an inherent risk of overfitting and anatomical evidence. Pang et al.1 put forth harmonic modes derived from the brains geometry as a previously underrecognized model to explain brain-wide dynamics. Their reconstruction framework relies on multiple linear regression to fit brain patterns using a basis set and then calculating Pearsons correlation between original data and fitted data, both parcellated using an atlas with 180 parcels in each hemisphere2. In addition to the overarching challenge of accepting any model as an actual reflection of the real world3, we here provide empirical results and theoretical arguments that highlight the need for further consideration regarding geometric eigenmodes as a model of macroscale brain activity.

neuroscience↗

bioRxiv2023

Decrypting the functional design of unmodified translation elongation factor P

Stalling of ribosomes during polypeptide synthesis due to consecutive proline motifs is a challenge faced by organisms across all kingdoms. To overcome this, bacteria employ translation elongation factor P (EF-P), while archaea and eukaryotes rely on a/eIF5A. Typically, these elongation factors become active only after undergoing post-translational modifications (PTMs) such as {beta}-lysinylation, (deoxy-)hypusinylation, rhamnosylation, or 5-aminopentanolyation. An exception to this rule is found in EF-P members of the PGKGP-subfamily, which remain unmodified. However, the mechanism behind the ability of certain bacteria to avoid metabolically and energetically costly PTMs, while retaining active EF-P, remains unclear. In this study, we investigated the design principles governing the full functionality of unmodified EF-Ps in Escherichia coli. We first screened for naturally unmodified EF-Ps that are active in an E. coli reporter strain. We identified EF-P from Rhodomicrobium vannielii capable of rescuing the growth deficiencies and changes in the proteome of an E. coli {Delta}epmA mutant lacking the gene for the modifying EF-P-(R)-{beta}-lysine ligase. We then identified specific amino acids in domain I of the unmodified EF-P variant that affected its activity. Ultimately, we transferred these functional properties to other marginally active members of the PGKGP EF-P subfamily, resulting in fully functional unmodified variants in E. coli. These results have implications for the improved heterologous expression of polyproline-containing proteins in E. coli and offer applications in other bacterial hosts. Understanding the mechanisms that underlie the functionality of unmodified EF-P provides insights into cellular adaptations to optimize protein synthesis.

microbiology↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2023

A fast-slow trait continuum at the level of entire communities

Across the tree of life, organismal functional strategies form a continuum from slow-to fast-growing organisms, in response to common drivers such as resource availability and disturbance. However, the synchronization of these strategies at the entire community level is untested. We combine trait data for >2800 above-and belowground taxa from 14 trophic guilds spanning a disturbance and resource availability gradient in German grasslands. Most guilds consistently respond to these drivers through both direct and trophically-mediated effects, resulting in a slow-fast axis at the level of the entire community. Fast trait communities were also associated with faster rates of whole ecosystem functioning. These findings demonstrate that slow and fast strategies can be manifested at the level of whole ecosystems, opening new avenues of ecosystem-level functional classification.

ecology↗

bioRxiv2023

Rat hepatitis E virus (HEV) cross-species infection and transmission in pigs

Rocahepevirus ratti, an emerging hepatitis E virus (HEV), has recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs cohoused with naive pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to cohoused animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism. Significance StatementNew zoonotic strains of Rocahepevirus ratti (rat HEV) have emerged infecting both immunocompetent and immunosuppressed people through unknown transmission sources. Pigs are a primary source of transmission for human HEV strains and could be serving a similar role for rat HEV transmission as rats are a common pest found on swine farms worldwide. Rats could be transmitting rat HEV to pigs which could then be transmitted to humans. Determining susceptibility of pigs to emerging zoonotic rat HEV strains can define potentially new transmission routes to inform public health policy and could provide pathology models for rat HEV disease.

microbiology↗

bioRxiv2023

Ribosome profiling reveals the fine-tuned response of Escherichia coli to mild and severe acid stress

The ability to respond to acidic environments is crucial for neutralophilic bacteria. Escherichia coli has a well-characterized regulatory network that triggers a multitude of defense mechanisms to counteract excess of protons. Nevertheless, systemic studies of the transcriptional and translational reprogramming of E. coli to different degrees of acid stress have not yet been performed. Here, we used ribosome profiling and RNA sequencing to compare the response of E. coli (pH 7.6) to sudden mild (pH 5.8) and severe near-lethal acid stress (pH 4.4) conditions that mimic passage through the gastrointestinal tract. We uncovered new differentially regulated genes and pathways, key transcriptional regulators, and 18 novel acid-induced candidate sORFs. By using machine learning leveraging large compendia of publicly available E. coli expression data, we were able to distinguish between the response to acid stress and general stress. These results expand the acid resistance network and provide new insights into the fine-tuned response of E. coli to mild and severe acid stress.

microbiology↗

bioRxiv2023

Urbanisation generates multiple trait syndromes for terrestrial taxa worldwide

Cities can host significant biological diversity. Yet, urbanisation leads to the loss of habitats and, potentially, to local extinctions. Understanding how multiple taxa respond to urbanisation globally is essential to promote and conserve biodiversity in cities and surrounding landscapes. Using a dataset with site-level occurrence and trait data of 5302 species from six terrestrial fauna taxonomic groups across 379 cities on 6 continents, we show that urbanisation produces taxon-specific changes in trait composition, with traits related to reproductive strategy consistently showing the strongest response. The effect of urbanisation on community trait composition is strongest at the largest spatial scale considered, and more closely linked to landscape composition (% urban) than arrangement (aggregation), although latitude and climatic variables remain a stronger influence. This study did not find evidence in support of a global urban taxa syndrome, but instead we suggest that there are four general urban trait syndromes, with resources associated with reproduction and diet likely to be driving patterns in traits associated with mobility and body size. Functional diversity measures showed a wide range of responses, leading to a shift in trait space that is most likely driven by the distribution and abundance of critical resources, and the urban trait syndrome displayed by individual species within a community. Further research is required to understand the interactions between the four general urban trait syndromes, resource distribution and abundance and changes in functional diversity of taxa at different spatial and temporal scales. Maximising opportunities to support species within taxa groups with different urban trait syndromes should be pivotal in conservation and management programmes within and among cities. This will reduce the likelihood of biotic homogenisation at the taxa level, and helps ensure that urban environments have the ecological capacity to respond to challenges such as climate change, further habitat fragmentation and loss, and other disruptions. These actions are critical if we are to reframe the role of cities in global biodiversity loss.

ecology↗

bioRxiv2023

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/537186v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@8ce1bdorg.highwire.dtl.DTLVardef@1385103org.highwire.dtl.DTLVardef@4ec496org.highwire.dtl.DTLVardef@14c9295_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIComplementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration. C_LIO_LIVEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis. C_LIO_LIIdentification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin. C_LIO_LIKDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered. C_LI

pathology↗

bioRxiv2023

An adaptive behavioral control motif mediated by cortical axo-axonic inhibition

Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.

neuroscience↗

bioRxiv2022

Intermediately Synchronised Brain States optimise trade-off between Subject Identifiability and Predictive Capacity

Functional connectivity (FC) refers to the statistical dependencies between activity of distinct brain areas. To study temporal fluctuations in FC within the duration of a functional magnetic resonance imaging (fMRI) scanning session, researchers have proposed the computation of an edge time series (ETS) and their derivatives. Evidence suggests that FC is driven by a few time points of high-amplitude co-fluctuation (HACF) in the ETS, which may also contribute disproportionately to interindividual differences. However, it remains unclear to what degree different time points actually contribute to brain-behaviour associations. Here, we systematically evaluate this question by assessing the predictive utility of FC estimates at different levels of co-fluctuation using machine learning (ML) approaches. We demonstrate that time points of lower and intermediate co-fluctuation levels provide overall highest subject specificity as well as highest predictive capacity of individual-level phenotypes.

neuroscience↗

bioRxiv2022

Genetic architecture of creativity and extensive genetic overlap with psychiatric disorders revealed from genome-wide association analyses of 241,736 individuals

Creativity is heritable and exhibits familial aggregation with psychiatric disorders, but its genomic basis and genetic relationship with psychiatric disorders remain largely unknown. Here, we conducted a genome-wide association study (GWAS) using an expanded, machine learning-based definition of creativity in individuals of European ancestry from the UK Biobank (n = 241,736) and identified 25 creativity-associated loci. Extensive genetic overlap with psychiatric disorders, including schizophrenia, major depression, bipolar I disorder, attention deficit/hyperactivity disorder, and anorexia nervosa, was demonstrated by the genetic correlation, polygenic risk score, and MiXeR analyses. The condFDR and conjFDR analyses identified additional loci for creativity and psychiatric disorders, as well as shared genetic loci between creativity and psychiatric disorders. This GWAS showed significant correlations with GWASs using traditional definitions of creativity and GWASs adjusted for educational attainment. Our findings contribute to the understanding of the genetic architecture of creativity and reveal its polygenic relationships with psychiatric disorders.

genomics↗

bioRxiv2022

Whole-brain dynamical modeling for classification of Parkinson's disease

Simulated whole-brain connectomes demonstrate an enhanced inter-individual variability depending on data processing and modeling approach. By considering the human brain connectome as an individualized attribute, we investigate how empirical and simulated whole-brain connectome-derived features can be utilized to classify patients with Parkinsons disease against healthy controls in light of varying data processing and model validation. To this end, we applied simulated blood oxygenation level-dependent signals derived by a whole-brain dynamical model simulating electrical signals of neuronal populations to reveal differences between patients and controls. In addition to the widely used model validation via fitting the dynamical model to empirical neuroimaging data, we invented a model validation against behavioral data, such as subject classes, which we refer to as behavioral model fitting and show that it can be beneficial for Parkinsonian patient classification. Furthermore, the results of machine-learning reported in this study also demonstrated that performance of the patient classification can be improved when the empirical data are complemented by the simulation results. We also showed that temporal filtering of blood oxygenation level-dependent signals influences the prediction results, where the filtering in the low-frequency band is advisable for Parkinsonian patient classification. In addition, composing the feature space of empirical and simulated data from multiple brain parcellation schemes provided complementary features that improve prediction performance. Based on our findings, we suggest including the simulation results with empirical data is effective for inter-individual research and its clinical application.

neuroscience↗

bioRxiv2022

Identification of two pyruvate transporters in Salmonella enterica serovar Typhimurium and their biological relevance

Pyruvate (CH3COCOOH) is the simplest of the alpha-keto acids and is at the interface of several metabolic pathways both in prokaryotes and eukaryotes. In an amino acid-rich environment, fast-growing bacteria excrete pyruvate instead of completely metabolizing it. The role of pyruvate uptake in pathological conditions is still unclear. In this study, we identified two pyruvate-specific transporters, BtsT and CstA, in Salmonella enterica serovar Typhimurium (S. Typhimurium). Expression of btsT is induced by the histidine kinase/response regulator system BtsS/BtsR upon sensing extracellular pyruvate (threshold 200 M), whereas expression of cstA is maximal in the stationary phase. Both pyruvate transporters were found to be important for the uptake of this compound, but also for chemotaxis to pyruvate, survival under oxidative and nitrosative stress, and persistence of S. Typhimurium in response to gentamicin. Compared with the wild-type, the {Delta}btsT{Delta}cstA mutant has disadvantages in antibiotic persistence in macrophages, as well as in colonization and systemic infection in gnotobiotic mice. These data demonstrate the surprising complexity of the two pyruvate uptake systems in S. Typhimurium.

microbiology↗

bioRxiv2021

Structural basis for mismatch surveillance by CRISPR/Cas9

The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016), the underlying molecular mechanisms by which Cas9 rejects or accepts mismatches are poorly understood (Kim et al., 2019; Liu et al., 2020; Slaymaker and Gaudelli, 2021). Here, we used kinetic analysis to guide cryo-EM structure determination of Cas9 at different stages of mismatch surveillance. We observe a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS), that occurs in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. We observe that highly tolerated PAM-distal mismatches achieve this kinked conformation through stabilization of a distorted duplex conformation via a flexible loop in the RuvC domain. Our results provide molecular insights into the underlying structural mechanisms that may facilitate off-target cleavage by Cas9 and provides a molecular blueprint for the design of next-generation high fidelity Cas9 variants that selectively reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.

biochemistry↗