Single-cell brain organoid screening identifies developmental defects in autism.

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Proteome-scale tagging and functional screening in mammalian cells by ORFtag.

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a promoter, peptide tag and splice donor to generate fusions between the tag and endogenous open reading frames (ORFs). We demonstrate the utility of ORFtag through functional screens for transcriptional activators, repressors and posttranscriptional regulators in mouse embryonic stem cells. Each screen recovers known and identifies new regulators, including long ORFs inaccessible by other methods. Among other hits, we find that Zfp574 is a highly selective transcriptional activator and that oncogenic fusions often function as transactivators.

A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection.

Stem cells intrinsically express a subset of genes which are normally associated with interferon stimulation and the innate immune response. However, the expression of these interferon-stimulated genes (ISG) in stem cells is independent from external stimuli such as viral infection. Here, we show that the interferon regulatory factor 1, Irf1, is directly controlled by the murine formative pluripotency gene regulatory network and transiently upregulated during the transition from naive to formative pluripotency. IRF1 binds to regulatory regions of a conserved set of ISGs and is required for their faithful expression upon exit from naive pluripotency. We show that in the absence of IRF1, cells exiting the naive pluripotent stem cell state are more susceptible to viral infection. Irf1 therefore acts as a link between the formative pluripotency network, regulation of innate immunity genes, and defense against viral infections during formative pluripotency.

Genomic surveillance of SARS-CoV-2 evolution by a centralised pipeline and weekly focused sequencing, Austria, January 2021 to March 2023.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

A reversible haploid mouse embryonic stem cell biobank resource for functional genomics.

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

The BTB-domain transcription factor ZBTB2 recruits chromatin remodelers and a histone chaperone during the exit from pluripotency.

Transcription factors (TFs) harboring broad-complex, tramtrack, and bric-a-brac (BTB) domains play important roles in development and disease. These BTB domains are thought to recruit transcriptional modulators to target DNA regions. However, a systematic molecular understanding of the mechanism of action of this TF family is lacking. Here, we identify the zinc finger BTB-TF Zbtb2 from a genetic screen for regulators of exit from pluripotency and demonstrate that its absence perturbs embryonic stem cell differentiation and the gene expression dynamics underlying peri-implantation development. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the BTB domain directly interacts with nucleosome remodeling and deacetylase and histone chaperone histone regulator A. Nucleosome remodeling and deacetylase recruitment is a common feature of BTB TFs, and based on phylogenetic analysis, we propose that this is a conserved evolutionary property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the BTB domain. Taken together, this study identifies a BTB-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition and defines unique and shared molecular functions of the BTB-domain TF family.

The histone chaperone CAF-1 safeguards somatic cell identity.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death.

Seventy-five genetic loci influencing the human red blood cell.

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer.

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-ß and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Loss of cohesin regulator PDS5A reveals repressive role of Polycomb loops.

Polycomb Repressive Complexes 1 and 2 (PRC1, PRC2) are conserved epigenetic regulators that promote transcriptional gene silencing. PRC1 and PRC2 converge on shared targets, catalyzing repressive histone modifications. Additionally, a subset of PRC1/PRC2 targets engage in long-range interactions whose functions in gene silencing are poorly understood. Using a CRISPR screen in mouse embryonic stem cells, we found that the cohesin regulator PDS5A links transcriptional silencing by Polycomb and 3D genome organization. PDS5A deletion impairs cohesin unloading and results in derepression of a subset of endogenous PRC1/PRC2 target genes. Importantly, derepression is not linked to loss of Polycomb chromatin domains. Instead, PDS5A removal causes aberrant cohesin activity leading to ectopic insulation sites, which disrupt the formation of ultra-long Polycomb loops. We show that these loops are important for robust silencing at a subset of PRC1/PRC2 target genes and that maintenance of cohesin-dependent genome architecture is critical for Polycomb regulation.

Ascl1 and Ngn2 convert mouse embryonic stem cells to neurons via functionally distinct paths.

Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.

Low-density lipoprotein receptor-related protein 1 (LRP1) as an auxiliary host factor for RNA viruses.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor-related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers.

ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.