RESUMO
Continual advancements in genomics have led to an ever-widening disparity between the rate of discovery of genetic variants and our current understanding of their functions and potential roles in disease. Systematic methods for phenotyping DNA variants are required to effectively translate genomics data into improved outcomes for patients with genetic diseases. To make the biggest impact, these approaches must be scalable and accurate, faithfully reflect disease biology, and define complex disease mechanisms. We compare current methods to analyze the function of variants in their endogenous DNA context using genome editing strategies, such as saturation genome editing, base editing and prime editing. We discuss how these technologies can be linked to high-content readouts to gain deep mechanistic insights into variant effects. Finally, we highlight key challenges that need to be addressed to bridge the genotype to phenotype gap, and ultimately improve the diagnosis and treatment of genetic diseases.
Assuntos
Edição de Genes , Variação Genética , Humanos , Edição de Genes/métodos , Variação Genética/genética , DNA/genética , Sistemas CRISPR-Cas/genética , Genômica/métodos , Animais , FenótipoRESUMO
Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.
Assuntos
Transporte Axonal , Axônios , Complexo Dinactina , Dineínas , Neurônios , Humanos , Complexo Dinactina/genética , Dineínas/genética , Células-Tronco NeuraisRESUMO
CRISPR screens with single-cell transcriptomic readouts are a valuable tool to understand the effect of genetic perturbations including single nucleotide variants (SNVs) associated with diseases. Interpretation of these data is currently limited as genotypes cannot be accurately inferred from guide RNA identity alone. scSNV-seq overcomes this limitation by coupling single-cell genotyping and transcriptomics of the same cells enabling accurate and high-throughput screening of SNVs. Analysis of variants across the JAK1 gene with scSNV-seq demonstrates the importance of determining the precise genetic perturbation and accurately classifies clinically observed missense variants into three functional categories: benign, loss of function, and separation of function.
Assuntos
Perfilação da Expressão Gênica , RNA Guia de Sistemas CRISPR-Cas , Genótipo , Transcriptoma , Nucleotídeos , Análise de Célula Única , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Variação Genética , Infecções por HIV , HIV-1 , Carga Viral , Humanos , Linhagem Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/genética , HIV-1/crescimento & desenvolvimento , HIV-1/fisiologia , Carga Viral/genética , África , Cromossomos Humanos Par 1/genética , Alelos , RNA Longo não Codificante/genética , Replicação ViralRESUMO
Understanding the effects of genetic variation in gene regulatory elements is crucial to interpreting genome function. This is particularly pertinent for the hundreds of thousands of disease-associated variants identified by GWAS, which frequently sit within gene regulatory elements but whose functional effects are often unknown. Current methods are limited in their scalability and ability to assay regulatory variants in their endogenous context, independently of other tightly linked variants. Here, we present a new medium-throughput screening system: genome engineering based interrogation of enhancers assay for transposase accessible chromatin (GenIE-ATAC), that measures the effect of individual variants on chromatin accessibility in their endogenous genomic and chromatin context. We employ this assay to screen for the effects of regulatory variants in human induced pluripotent stem cells, validating a subset of causal variants, and extend our software package (rgenie) to analyse these new data. We demonstrate that this methodology can be used to understand the impact of defined deletions and point mutations within transcription factor binding sites. We thus establish GenIE-ATAC as a method to screen for the effect of gene regulatory element variation, allowing identification and prioritisation of causal variants from GWAS for functional follow-up and understanding the mechanisms of regulatory element function.
Assuntos
Cromatina , Células-Tronco Pluripotentes Induzidas , Humanos , Cromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Ligação ProteicaRESUMO
CRISPR activation (CRISPRa) is an important tool to perturb transcription, but its effectiveness varies between target genes. We employ human pluripotent stem cells with thousands of randomly integrated barcoded reporters to assess epigenetic features that influence CRISPRa efficacy. Basal expression levels are influenced by genomic context and dramatically change during differentiation to neurons. Gene activation by dCas9-VPR is successful in most genomic contexts, including developmentally repressed regions, and activation level is anti-correlated with basal gene expression, whereas dCas9-p300 is ineffective in stem cells. Certain chromatin states, such as bivalent chromatin, are particularly sensitive to dCas9-VPR, whereas constitutive heterochromatin is less responsive. We validate these rules at endogenous genes and show that activation of certain genes elicits a change in the stem cell transcriptome, sometimes showing features of differentiated cells. Our data provide rules to predict CRISPRa outcome and highlight its utility to screen for factors driving stem cell differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Neurônios , Ativação Transcricional , Cromatina/genéticaRESUMO
Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.
Assuntos
Neoplasias Hematológicas , Neoplasias , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Edição de Genes , Neoplasias/genética , Mutação , Transdução de Sinais/genética , Sistemas CRISPR-CasRESUMO
The metabolic basis of Parkinson's disease pathology is poorly understood. However, the involvement of mitochondrial and endoplasmic reticulum stress in dopamine neurons in disease aetiology is well established. We looked at the effect of rotenone- and tunicamycin-induced mitochondrial and ER stress on the metabolism of wild type and microtubule-associated protein tau mutant dopamine neurons. Dopamine neurons derived from human isolated iPSCs were subjected to mitochondrial and ER stress using RT and TM, respectively. Comprehensive metabolite profiles were generated using a split phase extraction analysed by reversed phase lipidomics whilst the aqueous phase was measured using HILIC metabolomics. Mitochondrial and ER stress were both shown to cause significant dysregulation of metabolism with RT-induced stress producing a larger shift in the metabolic profile of both wild type and MAPT neurons. Detailed analysis showed that accumulation of triglycerides was a significant driver of metabolic dysregulation in response to both stresses in both genotypes. Whilst the consequence is similar, the mechanisms by which triglyceride accumulation occurs in dopamine neurons in response to mitochondrial and ER stress are very different. Thus, improving our understanding of how these mechanisms drive the observed triglyceride accumulation can potentially open up new therapeutic avenues.
RESUMO
There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimer's, Parkinson's, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Humanos , Microglia/metabolismo , Transcriptoma , Diferenciação Celular/genética , Doenças Neurodegenerativas/metabolismoRESUMO
We explored human induced pluripotent stem cells (hiPSCs) derived from different tissues to gain insights into genomic integrity at single-nucleotide resolution. We used genome sequencing data from two large hiPSC repositories involving 696 hiPSCs and daughter subclones. We find ultraviolet light (UV)-related damage in ~72% of skin fibroblast-derived hiPSCs (F-hiPSCs), occasionally resulting in substantial mutagenesis (up to 15 mutations per megabase). We demonstrate remarkable genomic heterogeneity between independent F-hiPSC clones derived during the same round of reprogramming due to oligoclonal fibroblast populations. In contrast, blood-derived hiPSCs (B-hiPSCs) had fewer mutations and no UV damage but a high prevalence of acquired BCOR mutations (26.9% of lines). We reveal strong selection pressure for BCOR mutations in F-hiPSCs and B-hiPSCs and provide evidence that they arise in vitro. Directed differentiation of hiPSCs and RNA sequencing showed that BCOR mutations have functional consequences. Our work strongly suggests that detailed nucleotide-resolution characterization is essential before using hiPSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Genômica , Humanos , Mutação , Nucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genéticaRESUMO
Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.
Assuntos
Células-Tronco Pluripotentes Induzidas , Mielopoese , Diferenciação Celular/genética , Linhagem da Célula/genética , Genômica , Hematopoese/genética , Humanos , Mielopoese/genéticaRESUMO
The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants. In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next-generation sequencing. We found that high levels of promoter methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific manner. In addition, we detected an over-representation of deletion events governed by a single 5'-terminal nucleotide at Cas9-induced DNA breaks. Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evidence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.
Assuntos
Sistemas CRISPR-Cas , Metilação de DNA , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Metilação de DNA/genética , Reparo do DNA , Edição de Genes , Mutação/genéticaRESUMO
(A)BACKGROUND: Musculoskeletal (MSK) first contact physiotherapy (FCP) is being rolled out in the National Health Service, but limited research exists on career pathways into MSK FCP, or on pre-and-post-registration educational preparation for the knowledge and skills that are required for musculoskeletal first contact physiotherapy. (B) OBJECTIVES: From the perspectives of existing MSK FCPs, the study sought to understand the pre-and-post-registration professional developmental journey into musculoskeletal first contact physiotherapy. (C) METHODS: Semi-structured interviews over the telephone were conducted with a self-selected and snowball sample of 15 MSK FCPs from across Britain. Framework analysis was used to analyze the interview transcripts. (D) RESULTS: Four overarching themes were identified: (1) Decision to choose a career path as a MSK FCP; (2) Relevancy of pre-registration physiotherapy (PT) education for MSK FCP; (3) Relevancy of post-registration continuing professional development for MSK FCP, and; (4) Improving pre-registration PT education for the foundational knowledge and skills required to work in musculoskeletal first contact physiotherapy. Each overarching theme generated several subthemes. (E)CONCLUSION: The research contributes to understanding the career pathway into the MSK FCP role and showed what relevant knowledge and skills were acquired for this role at pre-and-post registration levels. Findings will inform guidance for pre-registration PT curriculum development.
Assuntos
Doenças Musculoesqueléticas , Humanos , Doenças Musculoesqueléticas/terapia , Modalidades de Fisioterapia , Pesquisa Qualitativa , Medicina Estatal , TelefoneRESUMO
Young et al. examine the complexity of primary human microglia, and identify previously unknown cell states. Using expression quantitative trait locus (eQTL) mapping techniques, they identify 129 genes whose expression in microglia is linked to disease, and show that induced pluripotent stem cell (iPSC) models can be used for functional validation of common genetic mutations in microglia-associated diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Microglia , Mapeamento Cromossômico , Humanos , Microglia/metabolismo , Locos de Características Quantitativas/genéticaRESUMO
The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.
Assuntos
Anáfase/genética , Cromatina/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Humanos , Microscopia Intravital , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-LikeRESUMO
Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.
Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas/patologia , Fagocitose , Epitélio Pigmentado da Retina/patologia , Retinose Pigmentar/patologia , Diferenciação Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Mutação/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Epitélio Pigmentado da Retina/ultraestrutura , Retinose Pigmentar/genética , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismoRESUMO
Increasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to ß-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development.