Biology subjects

Wells, D.

Publications and source records attributed to Wells, D..

17 recordsLinked to original sources

bioRxiv2026

Conserved core RNAi machinery in trematode-vectoring snails indicates gene silencing potential in the absence of classical systemic and amplification effectors

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

evolutionary biology↗

bioRxiv2026

Chromosome-scale genome assembly of diploid halophyte Thinopyrum bessarabicum excludes J genome from polyploid Thinopyrum ancestry

Wild relatives of wheat harbour genetic diversity essential for improving resilience to climate-driven stresses, yet their deployment is hampered by unresolved evolutionary relationships and the absence of reference genomes. Here we present a chromosome-scale reference genome for Thinopyrum bessarabicum, a diploid halophyte and high-priority donor for wheat salt tolerance breeding. A key unresolved question is whether the diploid J genome contributed directly to the subgenome composition of extant polyploid Thinopyrum species, and which genomic features underpin its exceptional salt tolerance. Using this resource, we show that the diploid J genome of Th. bessarabicum is not represented among the subgenomes of polyploid Thinopyrum species, resolving a long-standing ambiguity in Triticeae genomics. Gene-level resolution of the reciprocal 4/5 chromosomal translocation across six related Triticeae species identifies conserved breakpoint gene pairs, supporting a single ancestral rearrangement. Genome-wide gene content analysis shows that halophytic capacity is underpinned by quantitative expansion of conserved stress-response gene families. Salt tolerance phenotyping validates chromosome 5J as a tolerance locus in both Th. bessarabicum and wheat introgression lines. A physically anchored marker framework and dual-reference skim-sequencing pipeline enable precise megabase-resolution characterisation of Th. bessarabicum introgressions in wheat, providing a genomic foundation for deploying J-genome diversity in crop improvement.

plant biology↗

bioRxiv2025

Serotonin signaling modulates growth and motility in juvenile Fasciola hepatica

Fasciola hepatica causes fasciolosis, a parasitic disease that poses significant animal and human health challenges. Control relies on flukicides, most of which are adulticides, with only triclabendazole effective against the pathogenic migratory juvenile. Classical neurotransmitter pathways are widely targeted by anthelmintics yet remain underexplored for flukicide development. Here we explore the importance of serotonin (5-HT) signaling in juvenile fluke. In silico analyses confirmed all F. hepatica life stages express a complete 5-HT signaling pathway encompassing genes encoding proteins for 5-HT synthesis, transport, and reuptake, as well as five putative 5-HT G protein-coupled receptors (GPCRs). Homology and binding motif analyses supported the presence of two 5-HT1 (Fh5HT1A, Fh5HT1B) and three 5-HT7 (Fh5HT7A, -7B, -7C) GPCRs. Immunocytochemistry and in situ hybridization revealed widespread neuronal expression of 5-HT, its synthetic enzyme tryptophan hydroxylase (FhTPH), and the GPCR Fh5HT7C. 5-HT addition stimulated juvenile fluke motility; consistent with this observation, serotonin reuptake inhibition, which causes 5-HT persistence at synaptic junctions, also enhanced juvenile movement. Silencing of FhTPH, a key enzyme in 5-HT synthesis, blunted juvenile motility, a phenotype reversed by the addition of 5-HT. Silencing the fluke vesicular monoamine transporter (FhVMAT), which packages 5-HT into synaptic vesicles, reduced juvenile motility, whilst silencing the 5-HT reuptake transporter (FhSERT) which recycles synaptic 5-HT increased juvenile motility and growth, consistent with 5-HT accumulation enhancing effects. Whilst combinatorial silencing of Fh5HT1 receptors reduced fluke motility, silencing Fh5HT7 receptors led to a greater reduction in motility. Exogenous addition of 5-HT partially rescued motility deficits of juveniles with silenced Fh5HT1 receptors, but 5-HT excitation was abolished in Fh5HT7-RNAi juveniles, exposing their importance to fluke motility. Notably, sustained 5-HT exposure promoted juvenile growth, but these effects were not blunted by receptor-RNAi. The findings emphasize a central role of serotonin signaling in both juvenile motility and growth, exposing novel aspects of receptor function and encouraging therapeutic exploitation for liver fluke control. Author SummaryThe liver fluke, Fasciola hepatica, causes fasciolosis, a neglected tropical disease that poses a significant burden on human and animal health. There is no vaccine for fasciolosis and treatment relies on a single drug, triclabendazole, to control the early stages of infection which cause liver pathology whilst migrating through the mammalian host. Single drug reliance has increased the incidence of drug resistance in both human and animal populations, such that there is a pressing need for the characterization of novel drug targets and development of new anthelmintics targeting liver fluke. The focus of this research is to examine the role of the serotonin signaling system of liver fluke, bridging a gap in knowledge to enable the exploitation of this signaling pathway for flatworm drug development. Here, bioinformatic analysis has characterized the pathway components and receptors in multiple clinically relevant flatworm parasite species. Chemical and functional genomic methods have been used to prove the integral function of serotonin in liver fluke biology, regulating motility and growth, both essential for parasite infection and survival. This work provides data that help validate the serotonergic system of liver fluke as a potential target for future anthelmintic development.

molecular biology↗

bioRxiv2025

Dietary variations drive divergent phenotypic, transcriptomic, and metatranscriptomic profiles in Biomphalaria glabrata, a schistosomiasis vector snail

BackgroundThe freshwater snail Biomphalaria glabrata is an important natural vector for the human parasitic trematode Schistosoma mansoni, which causes schistosomiasis. In the laboratory, B. glabrata are routinely maintained on simple lettuce diets. We aimed to explore and compare the impact of alternative diets on snail performance, global gene expression, and microbiome. MethodsSnails were raised in groups on fresh lettuce (FL), fish food (FF) and artificial snail gel (SG) diet for eight weeks, while measuring dietary impacts on growth, survival, and fecundity. RNA sequencing (RNA-Seq) was performed to correlate dietary phenotypes with changes in the snail transcriptome and associated microbial metatranscriptome. ResultsRelative to FL, FF and SG diets markedly enhanced growth, survival, and fecundity, with FF generating the highest fecundity rate. RNA-Seq identified 21,887 nutritionally modulated genes in the snail transcriptome. Fish food (FF) and SG diets drove upregulation of genes associated with antimicrobial immunity, growth, and reproduction, while elevated expression of genes linked to xenobiotic metabolism and oxidative stress was observed in FL-fed snails. Metatranscriptomic analysis identified 104 microbial classes, with a total of twenty-three classes significantly enriched in FF and SG snails, including short-chain fatty acid-producing and nutrient-cycling bacteria. Significant correlation (r = 0.63, p = 0.001) linked differentially expressed genes with enriched microbial taxa, highlighting the impact of diet on key snail health and performance metrics. ConclusionsThis work is the first nutritranscriptomic analysis of laboratory-bred B. glabrata. We describe key insights into the diet-phenotype-transcriptome-microbiome axis, which will inform dietary precision and optimisation for laboratory culture of B. glabrata. These data also highlight fundamental aspects of snail biology which could be exploited for molecular snail control approaches.

genomics↗

bioRxiv2025

Polo-like kinase 1 regulates growth in juvenile Fasciola hepatica

Fasciola sp. (liver fluke) are parasitic flatworms that impose a significant burden on the agri-food industry and human health. Immature worms can cause severe damage to the liver as they migrate towards the bile ducts, and yet there is only a single drug to treat this pathogenic life stage, driving the need to identify targets for novel flukicides. Given their crucial role in the growth/development of immature Fasciola hepatica, neoblast-like stem cells are an attractive source of new flukicide targets. Kinases are a hugely diverse group of phosphorylating enzymes with key roles in almost all cellular processes. Kinase dysregulation can result in the development of cancerous cells/tissues, linking many to cell cycle-associated proliferation and growth. Here, we annotate the F. hepatica kinome, identifying 271 putative protein kinases, representing around 1.6% of predicted F. hepatica protein-coding genes with family proportions similar to those of other parasitic flatworms. Many of these kinases, such as polo-like kinase 1 (fhplk1), are upregulated in immature worms undergoing rapid growth and development, a process underpinned by the proliferation of neoblast-like stem cells. RNA interference (RNAi)-mediated silencing of fhplk1 in juvenile liver fluke reduced growth and cell proliferation, suggesting a conserved role within the cell-cycle; the cessation of stem cell proliferation persisted for at least a week following fhplk1-RNAi. A PLK inhibitor (BI 2536) was shown to phenocopy the fhplk1-RNAi phenotype in a dose-dependent manner, supporting the feasibility of targeting F. hepatica neoblast-like cells through kinase inhibitors. Transcriptomic analysis of fhplk1-RNAi juveniles revealed 946 downregulated genes, principally associated with the cell cycle or ribosomes. Over 80 of these downregulated genes were also downregulated following juvenile F. hepatica irradiation, supporting roles for these kinases in neoblast-like stem cells, and marking them as putative targets for control. Among the 1244 upregulated genes in fhplk1-RNAi juvenile worms were many neurotransmitters, receptors and ion channels, exposing the apparent upregulation of diverse inter-cellular signalling systems. While many neurotransmitter pathways promote proliferation in mammalian systems the interaction between neoblast-like stem cells and neuronal signalling in parasitic flatworms remains elusive. Here, the transcriptomic response of fhplk1-RNAi juveniles supports a link between neoblast-like stem cell driven growth/development and neuronal signalling. Author SummaryThe liver fluke is a parasitic flatworm which causes disease in livestock and humans around the world. While establishing the infection, immature liver fluke migrate through the host liver causing significant damage. Unfortunately, only one drug is currently recommended for treatment of these immature worms, though resistance to this drug is now widespread and exposes the pressing need to develop novel drugs. As worms migrate through the liver, they undergo growth and development which is facilitated by proliferating stem cells. These neoblast-like cells are akin to stem cells seen in other organisms, and as such are responsible for new tissue growth. Many stem cells express kinases, a large family of enzymes that control many cellular processes. In this study we identified all the potential kinases in the liver fluke, including those that may function in neoblast-like stem cells. One of these kinases, polo-like kinase 1 (PLK1), has been linked to cancer development in humans. We used reverse genetics to silence this gene, allowing us to understand its function. Silencing PLK1 reduced worm growth and stopped neoblast-like cell proliferation, confirming that it has an important role in growth and cell division. Also, we showed that a PLK1 inhibitor, developed for cancer treatments, reduced growth and neoblast-like cell activity, illustrating the potential for targeting liver fluke kinases associated with neoblast-like cells with drugs, providing new routes to drug development. We then carried out RNA sequencing of worms after PLK1 was silenced to show the effects of neoblast-like cell loss on the expression of other genes. We found a decrease in the expression of genes that regulate cell division, but an increase in the expression of genes related to inter-cell signalling, including neuronal genes. This supports evidence for interactions between the nervous system and neoblast-like cells which could be exploited in future drug discovery.

molecular biology↗

bioRxiv2025

Genetic control of the leaf ionome in pearl millet and correlation with root and agromorphological traits

Pearl millet (Pennisetum glaucum) thrives in arid and nutrient-poor environments, establishing its role as a crucial cereal crop for food security in sub-Saharan Africa. Despite its remarkable adaptability, its yields remain below genetic potential, primarily due to limited water and nutrient availability. In this study, we conducted ionomic profiling and genome-wide association studies (GWAS) in field conditions across two growing seasons to unravel the genetic basis of nutrient acquisition in pearl millet. Soil ion content analyses revealed significant differences in nutrient distribution between field sites, while certain ions, such as phosphorus (P) and zinc (Zn), consistently displayed stratified accumulation patterns across years, suggesting stable depth-dependent trends. Evaluation of a genetically diverse panel of inbred lines revealed substantial variation in leaf ion concentrations, with high heritability estimates. Correlations between leaf ion content and root anatomical or agromorphological traits highlighted the intricate interplay between genetic and environmental factors shaping leaf ion accumulation. These analyses also uncovered potential trade-offs in nutrient acquisition strategies. GWAS identified genomic regions associated with leaf ion concentrations, and the integration of genetic and gene expression data facilitated the identification of candidate genes implicated in ion transport and homeostasis. Our findings provide valuable insights into the genetic regulation of nutrient acquisition in pearl millet, offering potential targets for breeding nutrient-efficient and climate-resilient varieties. This study underscores the importance of integrating genetic, physiological, and root architectural traits to enhance agricultural productivity and sustainability in resource-constrained environments.

plant biology↗

bioRxiv2023

Neoblast-like Stem Cells of Fasciola hepatica

The common liver fluke (Fasciola hepatica) causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of putative novel targets due to their role in development and homeostasis, including at host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, differentiation, and are sensitive to ionising radiation - all attributes of neoblasts in other flatworms. Changes in cell proliferation were also noted during the early stages of in vitro juvenile growth/development (around four to seven days post excystment), which coincided with a marked reduction in the nuclear area of proliferating cells. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologues associated with neoblast-like cells in Schistosoma mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblast-like cells and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics. Author SummaryLiver fluke are parasitic worms that infect both livestock and humans worldwide, threatening food security and human health. Treatments against this disease-causing parasite are limited, and growing resistance to drugs is undermining the effectiveness of control strategies. Since drugs represent the only viable control option, it is crucial that new drugs are discovered through the identification and validation of new drug targets. Stem cells play important roles in the normal growth and repair processes of many organisms, but when these cells become dysregulated through mutation, they can drive the development of cancers. Stem cells of liver fluke may be attractive novel drug targets as disruption would affect worm survival and/or development within their host. In this research we describe the characteristics of liver fluke stem cells, such as their sensitivity to radiation and their ability to develop into new cell types (key stem cell features). We used radiation in combination with RNA sequencing to identify genes associated with the liver fluke stem cells. Finally, we used reverse genetics to reduce the expression of a gene associated with stem cells, which led to the loss of stem cells and reduced worm growth/development. These data provide evidence to support the exploitation of stem cells as a source of novel drug targets for liver fluke control.

molecular biology↗

bioRxiv2022

Transcriptomic analysis reveals a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

Fasciola spp. liver fluke have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and then by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, notably fhe-let-7a-5p, fhe-mir-124-3p and, miRNAs predicted to target Wnt-signalling, supporting a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control. Author SummaryParasitic worms are notoriously difficult to study outside of a host organism. However, recent developments in culture methods for Fasciola hepatica liver fluke juveniles support growth and development of these parasites in the laboratory (in vitro) towards adult parasites. Having the ability to grow pathogenic juvenile stages in vitro enables functional studies to validate potential drug and vaccine targets. However, comparison of in vitro grown juveniles to juveniles retrieved from infected hosts (in vivo) shows considerable size differences suggesting at least some differences in biology that could undermine the relevance of data generated from in vitro maintained parasites. This study examines gene expression differences between in vitro and in vivo maintained F. hepatica juveniles via transcriptomic analysis to identify similarities and differences in their biology which may help explain differences in the rate of growth and development. 86% of genes were shown to be expressed at similar levels across treatment groups suggesting a high level of biological similarity between in vitro and in vivo juveniles. However, the genes that are expressed differently between these juveniles will help improve current culture methods and provide a new group of potential drug targets that impact on juvenile growth and development.

molecular biology↗

bioRxiv2021

Regional and clonal T cell dynamics at single cell resolution in immune checkpoint blockade

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of large and dual-expanded clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+ precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.

immunology↗

bioRxiv2021

Establishment of pan-Influenza A (H1-H18) and pan-Influenza B (pre-split, Vic/Yam) Pseudotype Libraries for efficient vaccine antigen selection

We have developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18, and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza serological and neutralization assays. We demonstrate its utility in detecting serum response to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further pre-clinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

immunology↗

bioRxiv2020

Integration in or Near Oncogenes Plays Only a Minor Role in Determining the in Vivo Distribution of HIV Integration Sites Before or During Suppressive Antiretroviral Therapy

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist. Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

microbiology↗

bioRxiv2020

Early Emergence and Long-Term Persistence of HIV-Infected T Cell Clones in Children

Little is known about the emergence and persistence of HIV-infected T cell clones in perinatally-infected children. We analyzed peripheral blood mononuclear cells for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8-17.4 months of age and with viremia suppressed for 6-9 years. We obtained 8,662 HIV-1 integration sites from pre-ART and 1,861 sites on ART. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6-9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMC infected ex-vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

microbiology↗

bioRxiv2020

Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 16% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

developmental biology↗

bioRxiv2019

ZCWPW1 is recruited to recombination hotspots by PRDM9, and is essential for meiotic double strand break repair

During meiosis, homologous chromosomes pair (synapse) and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, recombination initiates with double-strand breaks (DSBs) within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have yet been identified.\n\nWe identified Zcwpw1, which possesses H3K4me3 and H3K36me3 recognition domains, as highly co-expressed with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognizes CpG dinucleotides, including within many Alu transposons.\n\nMale Zcwpw1 homozygous knockout mice show completely normal DSB positioning, but persistent DMC1 foci at many hotspots, particularly those more strongly bound by PRDM9, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest a model where ZCWPW1 recognition of PRDM9-bound sites on either the homologous, or broken, chromosome is critical for synapsis, and hence fertility.\n\nGraphical Abstract LegendIn humans and other species, recombination is initiated by double strand breaks at sites bound by PRDM9. Upon binding, PRDM9 deposits the histone marks H3K4me3 and H3K36me, but the functional importance of these marks has remained unknown. Here, we show that PRDM9 recruits ZCWPW1, a reader of both these marks, to its binding sites genome-wide. ZCWPW1 does not help position the breaks themselves, but is essential for their downstream repair and chromosome pairing, and ultimately meiotic success and fertility in mice.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/821678v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@141deb9org.highwire.dtl.DTLVardef@18559deorg.highwire.dtl.DTLVardef@1cf380borg.highwire.dtl.DTLVardef@1689072_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2018

Unified single-cell analysis of testis gene regulation and pathology in 5 mouse strains

By removing the confounding factor of cellular heterogeneity, single cell genomics can revolutionize the study of development and disease, but methods are needed to simplify comparison among individuals. To develop such a framework, we assayed the transcriptome in 62,600 single cells from the testes of wildtype mice, and mice with gonadal defects due to disruption of the genes Mlh3, Hormad1, Cul4a or Cnp. The resulting expression atlas of distinct cell clusters revealed novel markers and new insights into testis gene regulation. By jointly analysing mutant and wildtype cells using a model-based factor analysis method, SDA, we decomposed our data into 46 components that identify novel meiotic gene regulatory programmes, mutant-specific pathological processes, and technical effects. Moreover, we identify, de novo, DNA sequence motifs associated with each component, and show that SDA can be used to impute expression values from single cell data. Analysis of SDA components also led us to identify a rare population of macrophages within the seminiferous tubules of Mlh3-/- and Hormad1-/- testes, an area typically associated with immune privilege. We provide a web application to enable interactive exploration of testis gene expression and components at http://www.stats.ox.ac.uk/~wells/testisAtlas.html

genomics↗

bioRxiv2017

Profiling G protein-coupled receptors of Fasciola hepatica identifies orphan rhodopsins unique to phylum Platyhelminthes

G protein-coupled receptors (GPCRs) are established drug targets. Despite their considerable appeal as targets for next-generation anthelmintics, poor understanding of their diversity and function in parasitic helminths has thwarted progress towards GPCR-targeted anti-parasite drugs. This study facilitates GPCR research in the liver fluke, Fasciola hepatica, by generating the first profile of GPCRs from the F. hepatica genome. Our dataset describes 146 high confidence GPCRs, representing the largest cohort of GPCRs, and the most complete set of in silico ligand-receptor predictions, yet reported in any parasitic helminth. All GPCRs fall within the established GRAFS nomenclature; comprising three glutamate, 135 rhodopsin, two adhesion, five frizzled and one smoothened GPCR. Stringent annotation pipelines identified 18 highly diverged rhodopsins in F. hepatica that maintained core rhodopsin signatures, but lacked significant similarity with non-flatworm sequences, providing a new sub-group of potential flukicide targets. These facilitated identification of a larger cohort of 76 related sequences from available flatworm genomes, representing new members of existing groups of flatworm-specific rhodopsins. These receptors imply flatworm specific GPCR functions, and/or co-evolution with unique flatworm ligands, and could facilitate development of exquisitely selective anthelminthics. Ligand binding domain sequence conservation relative to deorphanised rhodopsins enabled high confidence ligand-receptor matching of seventeen receptors activated by acetylcholine, neuropeptide F/Y, octopamine or serotonin. RNA-Seq analyses showed expression of 101 GPCRs across various developmental stages, with the majority expressed most highly in the pathogenic intra-mammalian juvenile parasites. These data identify a broad complement of GPCRs in F. hepatica, including rhodopsins likely to have key functions in neuromuscular control and sensory perception, as well as frizzled and adhesion families implicated, in other species, in growth, development and reproduction. This catalogue of liver fluke GPCRs provides a platform for new avenues into our understanding of flatworm biology and anthelmintic discovery.\n\nAuthor SummaryFasciola spp. liver fluke are important veterinary pathogens with impacts on human and animal health, and food security, around the world. Liver fluke have developed resistance to most of the drugs used to treat them (flukicides). Since no vaccines exist, we need to develop new flukicides as a matter of urgency. Most anthelmintic drugs used to treat parasitic worm infections operate by impeding the functioning of their nerve and muscle. In flatworms, most nervous signals are received by a type of receptor called a G protein-coupled receptor (GPCR). Since GPCRs control important parasite functions (e.g. movement, egg-laying, feeding), they represent appealing targets for new flukicides, but have not yet been targeted as such. This work exploited the F. hepatica genome to determine the quantity and diversity of GPCRs in liver fluke. We found more GPCRs in the Fasciola genome than have been reported in any other parasitic worm. These findings provide a foundation that for researchers to determine the functions of these receptors, and which molecules/ligands they are activated by. These data will pave the way to exploring the potential of F. hepatica GPCRs as targets for new flukicides.

bioinformatics↗

bioRxiv2017

Genome And Epigenome Engineering CRISPR Toolkit For Probing In Vivo cis-Regulatory Interactions In The Chicken Embryo

CRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish protocol for evaluation of mutational penetrance by targeted next generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.\n\nSummary StatementWe present an optimised toolkit for efficient genome and epigenome engineering using CRISPR in chicken embryos, with a particular focus on probing gene regulatory interactions during neural crest development.\n\nList of AbbreviationsGenome Engineering (GE), Epigenome Engineering (EGE), single guide RNA (sgRNA), Neural Crest (NC), Transcription Factor (TF), Next Generation Sequencing (NGS), somite stage (ss), Hamburger Hamilton (HH).

developmental biology↗