Biology subjects

Huang, W.

Publications and source records attributed to Huang, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Development of CYP8B1-specific inhibitors for metabolic diseases

Changes in bile acids (BAs) balance lead to various metabolic diseases, yet current treatment options targeting these changes remain severely limited. CYP8B1 (cytochrome P450, family 8, subfamily B, polypeptide 1) is a liver-exclusive enzyme that controls the synthesis of 12-hydroxylated (12-OH) BAs. Inhibiting CYP8B1 is an attractive therapeutic strategy for attenuating 12-OH BA levels, remodeling the gut microbiota and thereby ameliorating metabolic diseases. CYP8B1-specific inhibitors are currently lacking due to structural similarities between the active site of CYP8B1 and other CYPs, and the lack of suitable high-throughput screening (HTS) assays. Herein, we developed a highly sensitive, and specific enzyme-coupled fluorescence assay to detect CYP8B1 activity, making it ideal for HTS. Employing this assay, structure-based drug design by protein X-ray crystallography, structure-activity relationship (SAR) analyses, and in vitro and in vivo validation, we developed novel CYP8B1-specific small molecule inhibitors. The lead candidate alters BA composition, favorably remodels the gut microbiota, and corrects metabolic disease in a metabolic dysfunction-associated steatotic liver disease (MASLD) mouse model. These findings reveal that pharmacologically inhibiting CYP8B1 is able to reshape the BA and gut microbiota profiles and effectively ameliorates metabolic diseases. Overall, CYP8B1-specific inhibitors represent a first-in-class promising, safe and effective therapeutic strategy against metabolic disorders.

pharmacology and toxicology↗

bioRxiv2026

Spatial multi-omics reveals novel vascular adipocyte and B cell biology in human abdominal aortic aneurysm

Objective: Perivascular Adipose Tissue (PVAT), tertiary lymphoid structures (TLS), and the B lymphocytes they harbor have been implicated in regulating vascular diseases, including abdominal aortic aneurysm (AAA). Yet, the relationship of adipose tissue to TLS and B cell responses is poorly understood. Approach and Results: Here, we used a multi-omic approach to rigorously define the landscape of human AAA and characterize the adipocyte and TLS microenvironment with regard to B-cell responses to vascular antigens in human AAA and control non-aneurysmal abdominal aortas. Single-nucleus and single-cell RNA sequencing identified transcripts for lipogenesis, lipolysis, extracellular remodeling, haptotactic and chemotactic factors, with potential to recruit CD44+ leukocytes within AAAs. Immunohistology and Visium HD spatial transcriptomics revealed that at least two adipocyte types exist: one in the PVAT and another as isolated clusters in the medial-adventitial region in human AAAs but not in control aortas. Further characterization of these medial-adventitial adipocytes in the AAAs showed close association with TLSs. Mature TLSs recapitulated the organization of functional secondary lymphoid structures, with multiple B-cell follicles clustered to form a nodular structure partly covered by a layer of smooth muscle cells. The TLSs harbored isotype-switched B cells expressing IgG1, IgG3, or IgA, with dominant kappa chain usage and abundant IgG antibodies to aortic wall. Myeloid cells in AAA were implicated in promoting B cell effects, as they were the primary producers of B-cell activating factor (BAFF), a cytokine that promotes B cell infiltration, survival, and function. Conclusion: Altogether, our results support a novel model in which new adipocytes in the medial-adventitial layer produce factors that promote early TLS formation, and BAFF produced by myeloid cells promotes the survival and maturation of vascular wall-antigen-exposed B cells, leading to mature TLS formation, production of antibodies to AAA antigens, fueling inflammation and AAA growth.

immunology↗

bioRxiv2026

Human brain white matter charts in international populations

Brain charts provide normative references for identifying neurodevelopmental milestones and clinically meaningful deviations. Existing white matter (WM) charts are derived predominantly from European and North American populations, limiting generalizability. Here, we established the first set of population-specific WM charts of 198 morphological and microstructural phenotypes for both Chinese (N=19,215) and Western (N=15,584) populations across the human lifespan. Developmental trajectories and spatial organization of WM maturation and degeneration were broadly conserved across the populations, whereas maturational timing varied across pathways and metrics. The Chinese population showed earlier maturation in several projection and sensorimotor pathways, but more protracted maturation in higher-order association and limbic pathways. Chinese population-matched charts more accurately predicted WM phenotypes and detected more extreme disease-related deviations in brain health in the Chinese population than Western-derived models. Individual deviation patterns further discriminated patients from healthy controls and predicted cognitive performance and plasma biomarkers. Our findings reveal population-specific WM maturation and show that reference-population mismatch can reduce the accuracy and clinical sensitivity of normative brain charts.

neuroscience↗

bioRxiv2026

Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering

Selection-marker-assisted homology-directed repair (HDR) efficiently edits coding sequence termini but is poorly suited for internal exons. Here, we developed artificial reporter intron-based HDR (ARI-HDR), which inserts a selectable intron into target exons to enable precise internal-exon editing. Although designed for scarless removal by splicing, ARIs frequently exhibit inaccurate splicing. Using a random mutagenesis reporter assay, we identified intron upstream sequences (IUS) as key determinants of splicing fidelity and screened degenerate IUS variants to obtain reliably spliced ARI donors. ARI-HDR successfully introduced site-specific mutations into five human genes. Combining long single-stranded DNA donors with conversion-tract optimization increased editing efficiency to over 80%. This efficiency enabled 'Dirty-Editing,' a streamlined workflow that generates precisely edited mixed-cell populations without single-cell cloning. Together, ARI-HDR extends selection-based enrichment to internal exons across protein-coding genes, while our splicing-optimized intron design offers a broadly applicable solution for artificial intron-based technologies.

molecular biology↗

bioRxiv2026

Programmable design of functional RNAs through multimodal generative learning

RNA engineering is bottlenecked by the tight coupling among sequence, structure, and function, leaving the field reliant on heuristics or screening. We introduce RNAverse, a multimodal generative model that learns a conditionable design grammar from RNA sequences, experimental structural probing profiles, and functional annotations. Beyond setting a new state of the art across 16 predictive modeling tasks, RNAverse inverts into generative design across four distinct RNA classes through three regimes, requiring experimental testing of no more than 20 candidates per-task. Zero-shot in-painting generates snoRNAs that promote premature termination codon (PTC) readthrough, and chimeric aptazymes for ligand-controlled gene regulation, exceeding rational design or large-scale screens. In both settings, RNAverse outperforms family-adapted sequence-only baselines. Fine-tuned in-painting extends to an unseen functional class, yielding IGFBP3-binding aptamers with picomolar affinity that outperform SELEX winners. Iterative optimization engineers 5' UTRs that outperform clinical benchmarks, enhancing protein production in cell-based assays for both the Zolgensma gene therapy and the BNT162b2 COVID-19 vaccine, and boosting antibody responses to BNT162b2 in mice. Together, these results establish multimodal generative learning as a route from predictive RNA modeling to programmable specification of functional RNAs.

bioinformatics↗

bioRxiv2026

Towards Robust Automated Mouse Scratching Behavior Detection for Itch Research

Automated detection of mouse scratching behavior could reduce manual annotation and is essential for preclinical itch research. However, robust performance across laboratories remains a challenge for current methods. We present Scratch-AID+, a framework dedicated to automated mouse scratching detection, including a diverse and progressive set of approaches: classic ResNet-based detectors, task-adaptive fine-tuning of video foundation models, and in-context learning with advanced multimodal large language models. To simulate real-world use cases, we benchmark on a public dataset and utilize additional cross-laboratory video recordings for independent testing. We find that classic baselines suffered severe performance degradation on external data under the realistic setting with sparse scratching events. In contrast, fine-tuned video foundation models successfully bridged this generalizability gap, achieving significantly better detection performance. Furthermore, frozen multimodal large language models demonstrated promising out-of-the-box ability using only few-shot demonstrations. We also examined the effects of different video preprocessing strategies. Our findings highlight the improved performance and generalizability of the adapted foundation models for automated scratching behavior analysis and shed light on the real-world deployment of such detectors. Our code is publicly available at https://anonymous.4open.science/r/scratch aid-plus/.

bioinformatics↗

bioRxiv2026

Probing Physical Intuition in Large Language Models through Partner-Dependent Mutation Prediction

Structure-guided mutation design requires relating a proposed substitution to its molecular environment to anticipate its effects. Existing evaluations of large language models (LLMs) in protein engineering emphasize sequence-based prediction or tool-integrated workflows, leaving unclear whether LLMs themselves can infer mutation effects from structural evidence without dedicated predictors. We evaluate physical intuition-qualitative reasoning that connects structural observations with molecular interaction principles-using a benchmark that tests the same substitution in the same protein across different binding partners, with predictions based on local atomic coordinates and no dedicated mutation-effect predictors. In the tested comparisons, the best-performing LLM achieved group accuracy comparable to the evaluated energy predictors and Biomni, an LLM-driven tool workflow, while the methods differed in their prediction patterns. Most evaluated LLMs achieved higher group accuracy for larger affinity improvements. Structural interventions support selective dependence on mutation-site and partner information, while assessable structural claims largely agree with the supplied coordinates. These findings support direct LLM structural reasoning as a basis for developing rational mutation-design tools that connect molecular observations with partner-specific effects.

bioinformatics↗

bioRxiv2026

A conditional upper-bound design map for rhodopsin-augmented electro microbial CO2-to-biomass conversion

The value of rhodopsin proton pumps as light harvesters in electro-microbial CO2-to-biomass conversion depends on molecular photophysics, pathway energetics and reactor light delivery. We couple these scales for Cupriavidus necator, comparing Calvin-Benson-Bassham (CBB) and reductive glycine (rGlyP) assimilation. The model predicts a 17.9% dark rGlyP yield advantage, close to 16.5% measured in chemostats. At 568 nm, 100 PPFD and 45% membrane occupancy, light-derived ATP raises yield by 17% for rGlyP and 14% for CBB. Charging CO2 capture and formate electrosynthesis, a retinal-only reactor reaches 3.29% efficiency versus 2.56% in the dark. A carotenoid antenna raises this to 3.52% at the model-derived 70% transfer estimate and helps in 93.2% of uncertainty samples, but lowers efficiency at the thermodynamic limit by diverting photons from photovoltaics. Point-source CO2 cuts the relative light benefit from 38% to 8%. Rhodopsin is therefore most useful when upstream formate is energy-intensive, not as a general efficiency boost.

systems biology↗

bioRxiv2026

Inferring ecDNA Evolution from Allele Frequency Spectra

Extrachromosomal DNA (ecDNA) can arise early in the development of aggressive tumors or later upon progression, but the impact of chronology on patient outcome is not known. Currently, it is not possible to determine when an ecDNA arose in the course of tumor development and progression, based on genome sequencing data. Here, we develop an analytic method, ecEvol, to infer ecDNA emergence time, selection strength, mutation rate, and clonal dominance from allele frequency spectra in bulk whole-genome sequencing. EcEvol outperforms other methods and predicts parameters with high accuracy across a wide range of parameter values. Applying ecEvol to primary, treatment-naive tumors, we find that ecDNA typically emerges early and undergoes strong positive selection, albeit with substantial variation across cancer types. EcDNAs carrying known oncogenes emerge earlier and reach greater clonal dominance than ecDNAs lacking known oncogenes. In advanced and predominantly treated tumors, ecDNA emerges substantially later than in matched primary tumors, consistent with the generation and selection of new ecDNA during tumor progression and therapy. Longitudinal samples further support continued expansion of ecDNA-bearing lineages. Finally, earlier ecDNA emergence is associated with poorer overall survival. These results provide insight into the chronology of ecDNA formation from a single tumor biopsy taken at any point during the course of the disease, revealing that allele-frequency spectra encode the evolutionary history of ecDNA and provide a quantitative framework for reconstructing its emergence and expansion.

bioinformatics↗

bioRxiv2026

Mathematical Modelling of Stochastic Gene Expression Quantifies mRNA Heterogeneity in Cancer Cell Populations with Extrachromosomal DNA

Oncogene amplification on circular extrachromosomal DNA (ecDNA) has been linked to poor prognosis and higher treatment resistance in multiple types of human cancer. ecDNA are mobile genetic elements lacking centromeres that are partitioned unevenly into daughter cells at mitosis. While random segregation of ecDNA contributes to gene-copy-number heterogeneity among tumour cells, how ecDNA contribute to phenotypic heterogeneity, via mRNA and proteins, and how this affects targeted therapy outcomes is still not understood. In fact, cancer cell populations with ecDNA show remarkable heterogeneity in mRNA concentrations, which is not explained by gene-copy-number heterogeneity alone. In this paper, we use a stochastic model of gene transcription to mathematically model mRNA production and decay in cancer cells with ecDNA, extending a method that has been used to study phenotypic heterogeneity in eurokaryotic and prokaryotic cells. Using our model, we explore the space of possible mRNA distributions from a given ecDNA distribution, under different model parameter regimes. Our model is able to produce realistic mRNA distributions and we use it to estimate transcriptional parameters from single-cell measurements of ecDNA and mRNA counts in cell-line data. We discover a remarkable similarity in estimated parameters across cell lines and amplification types, indicating similar processes underlying transcription in ecDNA and chromosomal amplifications. We also model genotoxic therapy and use our model to predict the mRNA composition of persisting cells, showing the usefulness of such a modelling framework to understanding the origins of treatment resistance in ecDNA-containing cancers.

cancer biology↗

bioRxiv2026

Impaired astrocytic Cx43 syncytial network in lateral habenula affects synaptic connectivity and drives adolescent stress-induced depression

Adolescent depression is a major neuropsychiatric disorder induced by early life stress; the underlying cellular mechanisms instigating pathological circuit malformation remain unclear, and, consequently, effective therapeutic strategy is limited. Here, we analysed the contribution of astrocytes to early life stress-induced adolescent depression using a mouse model of parental separation. We identified a previously unrecognised regional specialisation of lateral habenula (LHb) astrocytes, characterised by compact cellular domains, abundant leaflets, and enhanced Cx43 gap junctional coupling. Parental separation selectively reduced astrocytic Cx43 expression and gap junctional communication in the LHb, which was accompanied by profound astrocytic atrophy and loss of leaflets. Single-cell sequencing further revealed that parental separation induces a broad reorganisation of LHb astrocyte transcriptional states, including metabolic, stress-response, and inflammatory programs; moreover, it specifically reduces astrocyte-neuron communication. Consistent with this, we found that structural remodelling of LHb astrocytes affected tripartite synapse geometry, resulting in impaired excitatory and inhibitory synaptic transmission, and neuronal Ca2+ hyperexcitability, all ultimately associated with depressive-like behaviours. Selective knockdown of astrocytic Cx43 in the LHb recapitulated these pathological changes and translated into depressive-like behaviours. Mechanistically, we found that impaired Cx43 gap junctional communication is associated with excessive astrocytic Ca2+ dynamics, resulting in Rac1-GTPase activation and decreased p-Ezrin levels. In vivo inhibition of LHb astrocytic Ca2+ signalling rescued astrocytic atrophy and depressive-like behaviours following parental separation. Together, our findings establish a Cx43-Ca2+-Rac1-p-Ezrin pathway that links gap junction malfunction to structural remodelling of astrocytes and disruption of neuron-astrocyte interactions in the developing LHb. Our study reveals astrocytic Ca2+ dysregulation as an active downstream effector of Cx43 dysfunction and identifies a regionally specialised astrocytic mechanism through which early-life stress disrupts synaptic transmission and neuronal function during adolescence, providing a potential cellular target for intervention in adolescent depression.

neuroscience↗

bioRxiv2026

A synthetic PGC1α co-activator reprograms microglial immunometabolism to preserve cone function in retinal degeneration

Retinitis pigmentosa (RP) is genetically heterogeneous, yet initiating mutations converge on primary rod degeneration followed by cone dysfunction and loss. Activated microglia are implicated in this secondary phase, but whether selective immunometabolic reprogramming of microglia preserves retinal function remains unknown. In human iPSC-derived microglia and retinal CD11b cells, PGC1 induction suppressed LPS-driven interferon signaling and cytokine release, yet Seahorse analysis and isotopologue tracing showed that energy metabolism remained altered. We therefore fused PGC1 to a multivalent p65-HSF1 module, yielding ~2.5-fold higher nuclear receptor output than native PGC1 and coordinated engagement of oxidative and anti-inflammatory programs in mouse retinal CD11b cells. Microglia-restricted expression in humanized RHOP347L mice preserved photopic responses through six months, increased retinal thickness and promoted ramified subretinal IBA1 morphology. Microglial immunometabolism thus represents a potentially genotype-independent neuroprotective target in RP, and engineered PGC1 co-activators provide a means to sustain oxidative metabolism under inflammatory pressure.

immunology↗

bioRxiv2026

Distinct synovial fluid B-cell differentiation and activation profiles in rheumatoid arthritis

CD11c+CD21- B cells are overrepresented in rheumatoid arthritis (RA). Here we delineate their functional state at site of inflammation and its molecular basis. Phenotypic profiling of paired blood and synovial fluid from patients with anti-citrullinated protein antibody (ACPA) seropositive RA (N=12), seronegative RA (N=10) and spondyloarthritis (N=8), demonstrated a striking enrichment of CD11c+CD21- B cells in synovial fluid in all three diseases (average of CD19+ cells: 67%, 49% and 51%). In ACPA-positive RA we identified increased proportions of CD11c+IgG+CD27low cells and several stages of pre-plasmablasts. The CD11c+CD21- cells had a phenotype indicating BCR activation, proliferation, and antigen presentation. This B-cell profile was mirrored by a LAG3+CTLA4+TIGIT+ peripheral helper T-cell program in ACPA-positive RA, contrasting a CCR6+ Th17-skewing in seronegative RA. Single-cell spatial proteomics revealed a unique CD11chighCD21 low B-cell surface architecture including a BCR co-receptor-tetraspanin signature with CD72-CD81 co-localization, which was elevated in RA. CD11c+ cells also displayed CD44-CD52/CD59 and MHCII-CD40/CD84 immune-modules absent in other B-cell subsets. In summary, CD11c+CD21- B cells are highly diverse, and while an activated antigen-experienced synovial profile was shared between disease groups, ACPA+ RA showed evidence of increased extrafollicular plasmablast differentiation. Surface receptor organization implicated a favoured BCR responsiveness, complement resistance and antigen presentation.

immunology↗

bioRxiv2026

Targeting ALCAT1 Ameliorates Cardiomyopathy in Barth Syndrome

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

cell biology↗

bioRxiv2026

Sub-stoichiometric modifications of Aplysia californica tRNAs and tRNA fragments revealed by integrating intact and bottom-up mass spectrometry

Transfer RNA (tRNA) modifications regulate protein synthesis, yet their sequence positions and stoichiometries are known only for a subset of tRNAs in model organisms. Aplysia californica is a key neurobiological model in which neuronal modified ribonucleosides have been linked to changes in animal behavior. However, Aplysia tRNA modifications have not been mapped, obscuring their roles in modulating neuronal functions. To address this knowledge gap, we leveraged hybridization pulldowns and an integrated mass spectrometry (MS) strategy combining nucleoside profiling, intact tRNA measurements, and bottom-up tRNA modification mapping to determine the full sequences of Aplysia tRNAGluCUC and tRNALysUUU. Sub-stoichiometric modifications including dihydrouridine (D), 5-methylcytidine (m5C), 5,2'-O-dimethyluridine (m5Um) and 1-methyladenosine (m1A) on tRNAGluCUC varied across tissues, suggesting tRNA modifications are regulated to accommodate translational needs. In addition to conserved thio-modifications in the anticodon loop of tRNALysUUU, we report 2-thiocytidine (s2C) at position 32, representing the first detection of s2C in eukaryotes. We also developed a generalizable method to directly detect tRNA halves by intact LC-MS, revealing endogenous tRNA cleavage patterns. Our findings illustrate the advantages of combining intact and bottom-up MS, reveal s2C as an unexpected tRNA modification in a classical neurobiological model and offer new insights into post-transcriptional regulation of animal physiology.

biochemistry↗

bioRxiv2026

Regulation of protein kinase pathways in primary neuron model of anti-NMDA receptor encephalitis.

The most common form of autoimmune encephalitis is associated with antibodies that target N-methyl-D-aspartic acid receptors (NMDARs). NMDARs play a pivotal role in neurotransmission and synaptic plasticity. Mounting evidence has shown that antibody targeting of the NMDAR GluN1 subunit, as in anti-NMDAR encephalitis, leads to NMDAR cross-linking and receptor internalization. However, the underlying signaling pathways affected by antibodies targeting NMDARs remain to be explored. We previously demonstrated that a human GluN1 monoclonal antibody 5F5 (GluN1 mAb 5F5) rapidly localizes to and regulates synaptic NMDAR function at native synapses of primary hippocampal neurons. Here, we sought to explore signaling targets of GluN1 mAb 5F5 in primary cortical neurons of either sex using subcellular fractionation, Western blotting, and label-free quantitative phosphoproteomics by mass spectrometry. We find that human GluN1 mAb 5F5 does not change NMDAR abundance or surface levels of GluN1 on primary cortical neurons at 2 hr. Despite this, we observe that GluN1 mAb 5F5 alters the phosphoproteome in synaptoneurosomes and regulates numerous synapse-related biological processes and protein kinase activities. Bioinformatic analyses suggest that these phosphoproteomic changes are positively correlated with NMDAR activation and negatively correlated with NMDAR inhibition. Together, these data suggest that GluN1 mAb 5F5 alters intracellular kinase signaling pathways in primary cortical neurons, likely by activating the NMDAR. These studies may help to identify novel therapeutic strategies for anti-NMDAR encephalitis and other antibody-mediated encephalitides targeting cell surface antigens.

neuroscience↗

bioRxiv2026

Additive genes contribute to yield heterosis in rice

The molecular mechanisms underlying heterosis remain unresolved, largely owing to the scarcity of accurately identified heterosis-associated genes. Here, we systematically investigate the inheritance patterns of three yield heterosis-related genes in rice, analyzing their effects in both homozygous and heterozygous genetic backgrounds. We find that heterozygosity at individual loci predominantly produces additive, rather than overdominant, effects. Nevertheless, heterozygous states of these genes generate yield heterosis in heterozygous genetic backgrounds, supporting the overdominance model. Our findings confirm that heterozygous additive genes are key contributors to yield heterosis in rice.

genetics↗

bioRxiv2026

CRISPR/Cas9 Optimization Using Electronic Genome Mapping: Potential Role for Studying Human Genetic Disease

CRISPR Cas9's ability to bind targeted and non-targeted sites and its functional interactions with other proteins and domains have been studied using many technologies. These studies are often limited by using oligos as substrates or by a reliance on predicted binding sites. We show how electronic genome mapping (EGM) with its ability to detect single-molecule DNA nicks can determine how gRNA mismatches impact targeted versus off-target binding across the whole human genome in vitro. Cas9 was found to be capable of binding a single site adjacent to the FXN repeat expansion responsible for Friedreich Ataxia using a gRNA specific for that sequence while gRNAs matching a repetitive sequence were able to bind hundreds of sites. Binding to perfectly matched gRNA sites was compared to sites with mismatches at each gRNA position. The PAM site and nearby seed region bases were confirmed as critical for binding. In contrast, single mismatches toward the gRNA 5' end allowed off-target binding. We also used EGM to determine how physically close the bound dCas9/gRNA complex can be relative to nearby DNA modification sites and still allow activity. Bound CRISPR/dCas9 inhibits nickases with recognition sites 4-7 bp away from the gRNA/PAM. Because EGM can simultaneously evaluate many mismatched off-target sites, these insights may be extended to other CRISPR systems and enable optimization of Cas proteins and gRNAs. This is the first example of EGM being used to characterize proteins and RNAs. This is also the first study demonstrating the combined application of CRISPR and EGM methodology and its potential role in the detection of specific repeat regions in the human genome that are associated with human repeat expansion disorders.

genomics↗