Biology subjects

Powell, J. E.

Publications and source records attributed to Powell, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

HLiCA: An integrated cell atlas of the healthy human liver

The human liver is composed of a heterogeneous mix of cell types. How these distinct populations contribute individually and collectively to liver function remains poorly understood. Although single-cell technologies have advanced our understanding of liver biology, individual studies have often been limited by small donor cohorts and inconsistent cell type annotations. Integrating multiple datasets can overcome these challenges and better capture biological variability. We present the Human Liver Cell Atlas (HLiCA), an integrated reference of non-disease liver cells assembled from eight datasets across six research centers, encompassing more than 525,000 cells from 110 donors. Developed in collaboration with the Human Cell Atlas Liver Bionetwork, the HLiCA incorporates expert-curated cell annotations refined through community feedback and dedicated cell type annotation meetings. The HLiCA classifies cells into six lineages and expands the cell type resolution to include 47 distinct cell types. Starting from raw sequencing reads, we realigned all data and performed rigorous benchmarking to ensure robust integration across technical and biological variables. Genetic ancestry was inferred for all samples to evaluate the range of ancestral backgrounds represented in the atlas. The expanded cell type annotation enabled identification of previously unrecognized liver cell types, including NRXN1+ stromal cells. Their presence was validated using spatial transcriptomics, which localized NRXN1+ stromal cells to periportal regions. With the number of donors included in the HLiCA we were able to examine cell type specific associations with demographic covariates. In hepatocytes, drug metabolism genes showed differential expression between sexes, and in cholangiocytes, mucus-production genes varied with age. As the largest and most genetically diverse human liver cell atlas to date, the HLiCA provides a comprehensive, well-annotated reference for the field, annotated by expert consensus. This resource will enable deeper interrogation of liver cellular diversity, architecture, and function in the healthy human liver and serve as a reference to understand changes that occur with disease.

genomics↗

bioRxiv2026

The diploid reference genome of a human embryonic stem cell line

Advances in DNA sequencing and assembly technologies are spurring a shift from haploid reference genomes to sample-specific diploid assemblies. Here, we generated the first telomere-to-telomere (T2T) diploid reference for the widely used human embryonic stem cell (hESC) line, H9 (WAe009-A). This haplotype-resolved assembly is highly accurate with comprehensive annotation of genes, segmental duplications, methylation, and chromatin conformation. Pangenomic and phased-locus inference point to H9s mixed ancestry with a predominant European component. H9-specific genomic features include near-perfect telomeres [~]1.65-fold longer than other T2T assemblies, consistent with telomerase activity during pluripotency; chromosome 17 inversions that can predispose offspring to neurological syndromes; and expansions of ncRNA clusters, with overall genomic stability maintained despite extensive culturing. Mapping multi-omic datasets to the genome, we demonstrate the power of this resource for allele-specific, high-precision transcriptomic, genetic, and epigenetic analyses, with far-reaching implications for human development and disease.

genomics↗

bioRxiv2025

Colibactin produced by a honeybee symbiont defends against pathogens and shapes the gut community

Colibactin is a bacterial genotoxin that is linked to colorectal cancer and implicated in interbacterial competition; however, its role in natural communities remains unknown. Frischella perrara, a symbiont living only in honeybee guts, produces colibactin and causes DNA damage. Here, we found that F. perrara mono-colonization reduces bee lifespan but increases survivorship following challenge with an opportunistic pathogen. F. perrara reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. clbS, a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but is absent from opportunistic colonizers including bee pathogens. Our findings provide evidence that colibactin functions in interbacterial competition, exerts selective pressure on community members, and confers protection against pathogens, while permitting co-evolved gut symbionts to persist.

microbiology↗

bioRxiv2025

A secreted endosymbiont protein essential for colonizing host cells

ABSTRACTIntracellular bacterial symbioses have arisen myriad times in eukaryotes, with dozens known from insects alone1,2. Beginning with Buchnera, the obligate endosymbiont of aphids, genomes of endosymbionts have illuminated their evolutionary origins and metabolic contributions to hosts3,4. However, the mechanisms by which nonculturable endosymbionts enter host cells and suppress cellular immune processes have remained unknown. We show that an uncharacterized Buchnera protein, here designated SyeA, was present in the Buchnera ancestor, is secreted and homologous to secreted effectors of bacterial pathogens and is essential for Buchnera transmission. Buchnera is transmitted via expulsion from specialized maternal cells and uptake by embryos5. Using immunofluorescence microscopy, we found that SyeA levels peak upon colonization, accompanied by actin accumulation at the entry site. SyeA localizes outside the host-derived membrane and actin layer surrounding each Buchnera cell and colocalizes in host cytoplasm with Rho1, which regulates actin polymerization. syeA knockdown disrupts colonization and embryonic development and elevates lysosomal activity, leading to Buchnera destruction6. Our findings provide rare insight into how an anciently associated, mutualistic endosymbiont achieves its intracellular existence. SyeA is a vestige of pathogenic origins followed by evolution of increased host control and erosion of the original, more complex pathogenicity machinery.

microbiology↗

bioRxiv2025

Mitochondrial Dysfunction in Endothelial Cells Drives Greater Vascular Impairment in Females with Diabetes-Associated Peripheral Artery Disease

BackgroundWomen with peripheral artery disease (PAD) experience poorer clinical outcomes than men, particularly in the setting of diabetes. However, the mechanistic basis for these sex- specific disparities remains unclear. MethodsHere, we investigated endothelial cell (EC) function(s) in diabetes-associated PAD, with a focus on sex differences. Limb tissues from patients with diabetes and chronic limb-threatening ischemia (CLTI) undergoing amputation, and a diabetes mouse model of hindlimb ischemia (HLI), were assessed for vasodilatory capacity, angiogenesis, oxidative stress and changes to expression of mitochondrial complex genes. ECs exposed to a hyperglycemic environment in vitro were assessed for mitochondrial function. The therapeutic potential of the mitochondrial-targeted antioxidant MitoQ was investigated. ResultsECs from females with diabetes-associated PAD have altered responses compared to males. Specifically, limb vessels and skeletal muscle from females exhibit reduced arterial relaxation, angiogenesis and increased oxidative stress in response to HLI in mice, and in tissues from patients. Single-cell RNA sequencing of murine limbs revealed marked suppression of EC mitochondrial complex genes in females with diabetes. Female human ECs exposed to high glucose had reduced respiration, reduced expression of mitochondrial genes and increased oxidative stress. Remarkably, MitoQ restored arterial relaxation and the angiogenic response in female diabetes- associated PAD. ConclusionOur findings uncover a striking sex-specific vulnerability involving oxidative stress and mitochondrial dysfunction in EC health in diabetes-associated PAD. These results highlight the need for sex-specific therapeutic strategies in diabetic PAD, which might include mitochondrial targeted antioxidant strategies.

pathology↗

bioRxiv2025

Endothelial Colony-Forming Cell Transcriptomic Profiling in CT-defined Coronary Artery Disease from the BioHEART-CT Study Implicate CCBE1 in Mitochondrial Dysfunction-associated Atherosclerosis

BackgroundEndothelial dysfunction is an early contributor to atherosclerosis. This study combined CT imaging of coronary artery disease (CAD) and patient-dervied endothelial colony-forming cells (ECFCs) transcriptional profiling to investigate potential mechanisms underlying endothelial dysfunction in atherosclerosis. MethodsTwenty-six individuals with CT-defined CAD and eighteen non-CAD controls were included in the Discovery Cohort for bulk RNA sequencing and transcriptomic analysis of ECFCs. Differential gene expression analysis was performed, and candidate genes were selected based on logFC and p-value. Candidate genes were carried forward for gene expression validation using quantitative real-time PCR (qRT-PCR) in a Validation Cohort. Mitochondrial reactive oxygen species (mROS) production and mitochondrial mass were assessed using multi-colour flow cytometry. Functional validation of the top candidate was conducted in using human umbilical vein endothelial cells (HUVECs) using loss-of-function genetic approach. Expression Quantitative Trait Loci (eQTL)-association analysis was conducted using genotype data from the BioHEART-CT cohort. ResultsPairwise analysis identified six differentially expressed protein-coding genes in CAD ECFCs: CCBE1 (Collagen and Calcium Binding EGF Domain-Containing 1), SPINT2, CRISPLD1, PIEZO2, EPB41L3, and AC005943.1. qRT-PCR in the Validation Cohort confirmed significantly higher CCBE1 expression in CAD patients. Individuals with relative CCBE1 fold change expression>10 had a 2.8-fold increase in the log-odds ratio of CT-defined CAD. CAD ECFCs displayed elevated mROS and mitochondrial mass. CCBE1 knockdown in HUVECs reduced mROS and mitochondrial mass without affecting proliferation or permeability, but shifted cells into a metabolically elevated state, marked by increased ATP production, respiration and glycolysis. CCBE1 cis-eQTLs were associated with increased odds of CAD in the BioHEART-CT cohort. ConclusionsCCBE1 expression in ECFCs was higher in patients with CT-defined CAD versus non-CAD. Quantitative assessment of transcript levels supported a causal relationship between greater CCBE1 expression and CAD burden and risk, and functional experiments on CCBE1 knockdown demonstrated improved mitochondrial function in human endothelial cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/670989v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@4e609aorg.highwire.dtl.DTLVardef@1a85810org.highwire.dtl.DTLVardef@12d97cdorg.highwire.dtl.DTLVardef@494ab3_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIThe endothelium plays a critical role in vascular health and susceptibility to atherosclerosis. C_LIO_LIMitochondrial dysfunction has been implicated in atherosclerosis, but its role and mechanism in individual susceptibility to CAD in humans is not known. C_LI What New Information Does This Article Contribute?O_LINovel approach integrating CT imaging with ECFC functional data to link vascular structure with endothelial biology ex vivo. C_LIO_LIPatients with CT-defined CAD had 3.6-fold higher CCBE1 expression compared to non-CAD within the Validation Cohort. C_LIO_LICis-eQTL-association analysis revealed increased odds of CAD. C_LIO_LICCBE1 knockdown improved mitochondrial function in human endothelial cells. C_LIO_LITogether, these 4 lines of evidence point to a novel and causal role for CCBE1 in human CAD. C_LI

cell biology↗

bioRxiv2025

The impact of sex on the immune system explored at the single-cell level

Sex has a key role in disease susceptibility, in particular autoimmunity. Sex differences of the immune system stems from genes and their interactions with intrinsic and external factors. However, the cellular-level factors influencing sexual dimorphism is not fully understood. We thus examined immune sex differences at single-cell resolution to dissect the genetic impacts. Female-biased sex-differentially expressed genes (DEGs) in multiple immune cells were involved in TNF[a] signalling, whereas male DEGs were enriched for ribosomal-related functions. While cis-eQTLs were less common on sex chromosomes, we identified over 1000 sex-specific eQTLs and 51 sex-interacting eQTLs on autosomes. When we examined the effect of genetic control on sex-DEGs, we found genetic variants affecting the female-biased expression FCGR3A in NK cells (rs2099684) and ITGB2 in Monocytes (rs760462), both of which are associated with systemic lupus erythematosus. Our work reveals novel biases masked in bulk analyses and highlights sexually dimorphic genes and pathways at baseline.

immunology↗

bioRxiv2025

Classification of indeterminate and off-target cell types within human kidney organoid differentiation

Human pluripotent stem cell-derived organoids are multicellular models of developing tissues proposed to recapitulate developmental stages of lineage commitment across time. Assessing how accurately protocols recapitulate development is challenged by the lack of accurate reference data sets for early human development, the presence of off-target states and the potential to form cellular states not present in vivo. This study addresses these challenges with respect to differentiation of human pluripotent stem cells to kidney organoids. Based on a factorial single-cell transcriptomic analysis across a 27-day differentiation protocol, including 150,957 cells collected at five time points, we present a comprehensive classification of predicted identity, representing an in vitro temporal transcriptional atlas. For early stages, cellular identity was defined in relation to existing human developmental data across time and organ system. As renal structures arose, this was coupled with the kidney-specific classification methods. In this way we identify predicted cellular states, predictable off-target cell types based on embryology and transitional mesodermal populations with no clear in vivo equivalent. An analysis of all existing single cell data on this same protocol suggests the presence of the same off-target trajectories irrespective of cell line or laboratory. This study provides a valuable resource to better identify and understand how off-target populations arise in vitro, benefiting the advancement of kidney organoid technologies towards therapeutic outcomes.

cell biology↗

bioRxiv2024

Multiscale analysis and functional validation of the cellular and genetic determinants of skeletal disease

Musculoskeletal diseases are a major health burden. Development of bone-active therapies has been hindered by limited understanding of the cells and genes that regulate the skeleton. We exploited the value of cross-species analysis and developed single-cell methodologies in skeletal tissues to define the critical endosteal compartment that regulates bone turnover. Thirty-four distinct cell types were identified, and disease-relevant cells prioritised by enrichment for rare skeletal disorder genes and bone mineral density-associated genes in an extended UK Biobank GWAS. Functional validation was undertaken in over one thousand genetically modified mouse models. Endothelial and vascular smooth muscle cells were identified as novel skeletal disease-relevant cells alongside osteoblast, chondrocyte and osteoclast cell lineages. Hundreds of cell-specific genes with unappreciated roles in skeletal pathophysiology were identified. This comprehensive cellular and molecular framework underpins skeletal physiology and disease, and will help prioritise new therapeutic targets to accelerate development of novel therapies to treat musculoskeletal disease.

genetics↗

bioRxiv2024

Single-cell atlas of the human immune system reveals sex-specific dynamics of immunosenescence

Immunosenescence, or immune aging, is characterized by both changes in cell type abundance and a decline in cellular function, leading to increased susceptibility to immune-related diseases. Yet, the extent to which sex influences the dynamic composition of immune cells during immunosenescence remains unknown. Here, we use single-cell RNA sequencing in peripheral blood mononuclear cells from 982 donors to uncover the sex-specific immune aging dynamics. We reveal that aging drives sexually dimorphic compositional and transcriptional shifts, with females showing stronger immune remodeling. Female-specific shifts include the expansion of three cytotoxic CD8+ T effector memory subpopulations and inflammatory monocytes. In addition, female CD4+ central memory T cells exhibit abundance shifts in subpopulations involved in autoimmunity alongside age-associated transcriptional signatures enriched for autoimmune-related pathways. Conversely, males show an age-related expansion of a B cell subpopulation associated with an asymptomatic precursor state to chronic lymphocytic leukemia. These sex-biased, functionally distinct immune subpopulations represent sex-specific hallmarks of immune aging. Our work underscores the complexity and sexual dimorphism of immunosenescence and supports the development of sex-specific strategies to promote healthy aging. Highlights- A single-cell transcriptomic atlas of >1 million PBMCs from 982 donors uncovers sex-specific immune aging trajectories. - Aging females show greater cellular remodeling and stronger transcriptional shifts than males. - Female-specific expansions with age of cytotoxic CD8+ T effector memory cells, inflammatory monocytes, and CD4+ T helper 22 cells reflect a systemic shift toward a pro-inflammatory, self-reactive immune state. - Male-specific B cell shifts reveal expansion of CD5+ clonal subpopulations linked to leukemic precursor states. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/622096v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@161b6b1org.highwire.dtl.DTLVardef@214d55org.highwire.dtl.DTLVardef@5c3ee8org.highwire.dtl.DTLVardef@263227_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Genetic variants associated with cell-type-specific intra-individual gene expression variability reveal new mechanisms of genome regulation

Gene expression levels can vary substantially across cells, even in a seemingly homogeneous cell population. Identifying the relationships between genetic variation and gene expression is critical for understanding the mechanisms of genome regulation. However, the genetic control of gene expression variability among the cells within individuals has yet to be extensively examined. This is primarily due to the statistical challenges, such as the need for sufficiently powered cohorts and adjusting mean-variance dependence. Here, we introduce MEOTIVE (Mapping genetic Effects On inTra-Individual Variability of gene Expression), a novel statistical framework to identify genetic effects on the gene expression variability (sc-veQTL) accounting for the mean-variance dependence. Using single-cell RNA-seq data of 1.2 million peripheral blood mononuclear cells from 980 human donors, we identified 14 - 3,488 genes with significant sc-veQTLs (study-wide q-value < 0.05) across different blood cell types, 2,103 of which were shared across more than one cell type. We further detected 55 SNP-gene pairs (in 34 unique genes) by directly linking genetic variations with gene expression dispersion (sc-deQTL) regardless of mean-variance dependence, and these genes were enriched in biological processes relevant to immune response and viral infection. An example is rs1131017 (p<9.08x10-52), a sc-veQTL in the 5UTR of RPS26, which shows a ubiquitous dispersion effect across cell types, with higher dispersion levels associated with lower auto-immune disease risk, including rheumatoid arthritis and type 1 diabetes. Another example is LYZ, which is associated with antibacterial activity against bacterial species and was only detected with a monocyte-specific deQTL (rs1384) located at the 3 UTR region (p=1.48x10-11) and replicated in an independent cohort. Our results demonstrate an efficient and robust statistical method to identify genetic effects on gene expression variability and how these associations and their involved pathways confer auto-immune disease risk. This analytical framework provides a new approach to unravelling the genetic regulation of gene expression at the single-cell resolution, advancing our understanding of complex biological processes.

genomics↗

bioRxiv2023

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

bioengineering↗

bioRxiv2023

Identifying genetic variants that influence the abundance of cell states in single-cell data

Introductory ParagraphTo understand genetic mechanisms driving disease, it is essential but difficult to map how risk alleles affect the composition of cells present in the body. Single-cell profiling quantifies granular information about tissues, but variant-associated cell states may reflect diverse combinations of the profiled cell features that are challenging to predefine. We introduce GeNA (Genotype-Neighborhood Associations), a statistical tool to identify cell state abundance quantitative trait loci (csaQTLs) in high-dimensional single-cell datasets. Instead of testing associations to predefined cell states, GeNA flexibly identifies the cell states whose abundance is most associated with genetic variants. In a genome-wide survey of scRNA-seq peripheral blood profiling from 969 individuals,1 GeNA identifies five independent loci associated with shifts in the relative abundance of immune cell states. For example, rs3003-T (p=1.96x10-11) associates with increased abundance of NK cells expressing TNF- response programs. This csaQTL colocalizes with increased risk for psoriasis, an autoimmune disease that responds to anti-TNF treatments. Flexibly characterizing csaQTLs for granular cell states may help illuminate how genetic background alters cellular composition to confer disease risk.

genetics↗

bioRxiv2023

Mitigating a TDP-43 proteinopathy using RNA-targeting CRISPR effector proteins

The TDP-43 proteinopathies, which include amyotrophic lateral sclerosis and frontotemporal dementia, are a devastating group of neurodegenerative disorders that are characterized by the mislocalization and aggregation of TDP-43. Here we demonstrate that RNA-targeting CRISPR effector proteins, a programmable class of gene silencing agents that includes the Cas13 family of enzymes and Cas7-11, can be used to mitigate TDP-43 pathology when programmed to target ataxin-2, a modifier of TDP-43-associated toxicity. In addition to inhibiting the aggregation and transit of TDP-43 to stress granules, we find that the in vivo delivery of an ataxin-2-targeting Cas13 system to a mouse model of TDP-43 proteinopathy improved functional deficits, extended survival, and reduced the severity of neuropathological hallmarks. Further, we benchmark RNA-targeting CRISPR platforms against ataxin-2 and find that high-fidelity forms of Cas13 possess improved transcriptome-wide specificity compared to Cas7-11 and a first-generation effector. Our results demonstrate the potential of CRISPR technology for TDP-43 proteinopathies.

bioengineering↗

bioRxiv2022

An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.

genomics↗

bioRxiv2022

Multimodal single cell analysis of the paediatric lower airway reveals novel immune cell phenotypes in early life health and disease

Inflammation is a key driver of cystic fibrosis (CF) lung disease, not addressed by current standard care. Improved understanding of the mechanisms leading to aberrant inflammation may assist the development of effective anti-inflammatory therapy. Single-cell RNA sequencing (scRNA-seq) allows profiling of cell composition and function at previously unprecedented resolution. Herein, we seek to use multimodal single-cell analysis to comprehensively define immune cell phenotypes, proportions and functional characteristics in preschool children with CF. We analyzed 42,658 cells from bronchoalveolar lavage of 11 preschool children with CF and a healthy control using scRNA-seq and parallel assessment of 154 cell surface proteins. Validation of cell types identified by scRNA-seq was achieved by assessment of samples by spectral flow cytometry. Analysis of transcriptome expression and cell surface protein expression, combined with functional pathway analysis, revealed 41 immune and epithelial cell populations in BAL. Spectral flow cytometry analysis of over 256,000 cells from a subset of the same patients revealed high correlation in major cell type proportions across the two technologies. Macrophages consisted of 13 functionally distinct sub populations, including previously undescribed populations enriched for markers of vesicle production and regulatory/repair functions. Other novel cell populations included CD4 T cells expressing inflammatory IFN/{beta} and NF{kappa}B signalling genes. Our work provides a comprehensive cellular analysis of the pediatric lower airway in preschool children with CF, reveals novel cell types and provides a reference for investigation of inflammation in early life CF.

systems biology↗

bioRxiv2022

CRISPR base editing of cis-regulatory elements enables target gene perturbations

CRISPR technology has demonstrated broad utility for controlling target gene expression; however, there remains a need for strategies capable of modulating expression via the precise editing of non-coding regulatory elements. Here we demonstrate that CRISPR base editors, a class of gene-modifying proteins capable of creating single-base substitutions in DNA, can be used to perturb gene expression via their targeted mutagenesis of cis-acting sequences. Using the promoter region of the human huntingtin (HTT) gene as an initial target, we show that editing of the binding site for the transcription factor NF-{kappa}B led to a marked reduction in HTT gene expression in base-edited cell populations. We found that these gene perturbations were persistent and specific, as a transcriptome-wide RNA analysis revealed minimal off-target effects resulting from the action of the base editor protein. We further demonstrate that this base-editing platform could influence gene expression in vivo as its delivery to a mouse model of Huntingtons disease led to a potent decrease in HTT mRNA in striatal neurons. Finally, to illustrate the applicability of this concept, we target the amyloid precursor protein, showing that multiplex editing of its promoter region significantly perturbed its expression. These findings demonstrate the potential for base editors to regulate target gene expression.

bioengineering↗

bioRxiv2022

Honey bee functional genomics using symbiont-mediated RNAi

Bees are indispensable pollinators and model organisms for studying social behavior, development, and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a host RNA interference response that reduces expression of a targeted gene. To employ this FUGUES (FUnctional Genomics Using Engineered Symbionts) procedure, a double-stranded RNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by five days after colonization. This protocol can be accomplished in four weeks by bee researchers with microbiology and molecular cloning skills.

genetics↗