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Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. Blood stem cells and blood progenitor cells replenish a person’s entire blood system throughout their life and are crucial for survival. The stem cells have the potential to become any type of blood cell – including white blood cells and red blood cells – while the progenitor cells are slightly more restricted in the types of blood cell they can become. It is important to understand how the balance of cell types is maintained because, in cancers of the blood (also known as leukaemias), this organisation is lost and some cells proliferate abnormally. Almost all of a person’s cells will contain the same genetic information, but different cell types arise when different genes are switched on or off. The genes encoding proteins called transcription factors are particularly important because the proteins can control – either by activating or repressing – many other genes. Importantly, some of these genes will encode other transcription factors, meaning that these proteins essentially work together in networks. Schütte et al. have now combined extensive biochemical experiments with computational modelling to study some of the transcription factors that define blood stem cells and blood progenitor cells in mice. Firstly, nine transcription factors, which were already known to be important in blood stem cells, were thoroughly studied in mouse cells that could be grown in the laboratory. These experiments provided an overall view of which other genes these transcription factors control. Additional targeted investigations of the nine transcription factors then revealed how these proteins act in combination to activate or repress their respective activities. With this information, Schütte et al. built a computational model, which accurately reproduced how real mouse blood stem and progenitor cells behave when, for example, a transcription factor is deleted. Furthermore, the model could also predict what happens in single cells if the amounts of the transcription factors change. Lastly, Schütte et al. studied a common type of leukaemia. The model showed that the mutations that occur in this cancer change the finely tuned balance of the nine transcription factors; this may explain why leukaemia cells behave abnormally. In future these models could be extended to more transcription factors and other cell types and cancers.

Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf . Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.

Human cerebellar development is unique and cannot be fully replicated in animal models. Although human stem cell-derived cerebellar organoid models are increasingly being applied to model cerebellar diseases, their potential to provide insight into normal human cerebellar development remains underexplored. Here, we used CRISPR-based gene editing in cerebellar organoids as an approach for modelling specific features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We generated a fluorescent FOXP2 reporter line in human induced pluripotent stem cells to enable the characterisation of FOXP2-expressing cells during cerebellar organoid differentiation. Through transcriptomic profiling of FOXP2 reporter cerebellar organoids and cross-referencing with existing cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders.

HERV-K HML-2 (HK2) has been proliferating in the germ line of humans at least as recently as 250,000 years ago, with some integrations that remain polymorphic in the modern human population. One of the solitary HK2 LTR polymorphic integrations lies between exons 17 and 18 of RASGRF2, a gene that affects dopaminergic activity and is thus related to addiction. Here we show that this antisense HK2 integration (namely RASGRF2-int) is found more frequently in persons who inject drugs compared with the general population. In a Greek HIV-1–positive population (n = 202), we found RASGRF2-int 2.5 times (14 versus 6%) more frequently in patients infected through i.v. drug use compared with other transmission route controls (P = 0.03). Independently, in a United Kingdom-based hepatitis C virus-positive population (n = 184), we found RASGRF2-int 3.6 times (34 versus 9.5%) more frequently in patients infected during chronic drug abuse compared with controls (P < 0.001). We then tested whether RASGRF2-int could be mechanistically responsible for this association by modulating transcription of RASGRF2. We show that the CRISPR/Cas9-mediated insertion of HK2 in HEK293 cells in the exact RASGRF2 intronic position found in the population resulted in significant transcriptional and phenotypic changes. We also explored mechanistic features of other intronic HK2 integrations and show that HK2 LTRs can be responsible for generation of cis-natural antisense transcripts, which could interfere with the transcription of nearby genes. Our findings suggest that RASGRF2-int is a strong candidate for dopaminergic manipulation, and emphasize the importance of accurate mapping of neglected HERV polymorphisms in human genomic studies.

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the role of lipid metabolism to generate them remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified cholesteryl ester-enriched lipid droplets (CE-LDs) as critical host components required for flavivirus infection. CE-LD formation is mediated by Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2), whose activities were upregulated early during infection, coinciding with increased CE-LD formation and transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic ∼100-fold reduction in viral production. Mechanistically, CE-LDs display distinct proteomic signatures, enriched in fatty acid remodelling enzymes, GTPases, and lipid transport proteins. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. Disrupting CE-LD formation via SOAT1/2-deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the physiological and clinical relevance of viral LD exploitation, analysis in iPSC-derived macrophages mirrored findings in Huh7 cells, and dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever. This study establishes CE-LDs as essential host metabolic hubs that enable flavivirus morphogenesis and identifies host LD metabolism as a promising therapeutic target for combating flavivirus infections.

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the role of lipid metabolism to generate them remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified cholesteryl ester-enriched lipid droplets (CE-LDs) as critical host components required for flavivirus infection. CE-LD formation is mediated by Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2), whose activities were upregulated early during infection, coinciding with increased CE-LD formation and transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic ~100-fold reduction in viral production. Mechanistically, CE-LDs display distinct proteomic signatures, enriched in fatty acid remodelling enzymes, GTPases, and lipid transport proteins. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. Disrupting CE-LD formation via SOAT1/2-deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the physiological and clinical relevance of viral LD exploitation, analysis in iPSC-derived macrophages mirrored findings in Huh7 cells, and dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever. This study establishes CE-LDs as essential host metabolic hubs that enable flavivirus morphogenesis and identifies host LD metabolism as a promising therapeutic target for combating flavivirus infections.

Patched 1 (PTCH1) is the primary receptor for Sonic Hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma (MB). As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we employ cerebellar organoids differentiated from human induced pluripotent stem cells (iPSC) combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings support the occurrence of developmental mechanisms in cerebellar organoids that mirror in vivo processes of regionalisation and SHH signalling, and offer new insight into early pathophysiological events of MB tumorigenesis. Differentiation of human iPSC into cerebellar organoids Homozygous LOF of PTCH1 prevents cerebellar organoid differentiation PTCH1+/- cerebellar organoids display tissue-specific effects of SHH signalling Early altered gene expression relevant for MB in PTCH1+/- cerebellar organoids

In this study, we demonstrate the potential of cerebellar organoids for studying features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We used CRISPR gene editing in human iPSCs to generate a fluorescent FOXP2-reporter line. By combining transcriptomic analysis of iPSC-derived cerebellar organoids with published cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders. Our study demonstrates the power of cerebellar organoids to model early human developmental processes and disorders.Competing Interest StatementThe authors have declared no competing interest.