RESUMEN
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.
Asunto(s)
Alelos , Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/genética , Animales , Benchmarking , Proteína 9 Asociada a CRISPR/genética , Conjuntos de Datos como Asunto , Humanos , Ratones , MutaciónRESUMEN
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.
Asunto(s)
Bancos de Muestras Biológicas , Genómica/métodos , Haploidia , Células Madre Embrionarias de Ratones/metabolismo , Mutación , Animales , Vasos Sanguíneos/citología , Linaje de la Célula/genética , Resfriado Común/genética , Resfriado Común/virología , Genes Esenciales/genética , Pruebas Genéticas , Células HEK293 , Homocigoto , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Neovascularización Fisiológica/genética , Fosfolipasas A2 Calcio-Independiente/genética , Fosfolipasas A2 Calcio-Independiente/metabolismo , Rhinovirus/patogenicidadRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas/genética , Linaje de la Célula/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , ARN Guía de Kinetoplastida , Análisis de la Célula Individual/métodos , Animales , Células Cultivadas , Fibroblastos/citología , Técnicas de Inactivación de Genes , Vectores Genéticos , Ratones , Células Madre Pluripotentes/citología , Retroviridae/genética , Relación Señal-RuidoRESUMEN
Natural compounds constitute a major resource for the development of medicines for multiple diseases. While many natural compounds show strong biological activity, the mechanisms that confer clinical benefits are often elusive and have been attributed to multiple pathways. Periplogenin (PPG), a natural compound isolated from Cortex periplocae, exhibits strong anti-tumor activities in several human cancer cell lines. However, its molecular mode of action remained unclear. In this study, we leveraged a forward genetic screening approach in DU145 prostate cancer cells to uncover the molecular target of PPG using chemical mutagenesis. Next generation sequencing revealed that a single amino acid substitution at amino acid 804 in ATP1A1 (ATPase Na + /K + Transporting Subunit Alpha 1) confers resistance to the cytotoxic activity of PPG. Mechanistically, ATP1A1 T804 forms a hydrogen bond with PPG which is abolished by the T804A substitution in ATP1A1, resulting in resistance to PPG treatment in vitro. Importantly, in vivo, PPG strongly suppressed tumor development in a DU145 xenograft model whereas DU145 xenograft tumors carrying a ATP1A1-T804A mutation were largely unaffected by the treatment. These findings demonstrate that PPG suppresses the growth of DU145 prostate cancer cells in vitro and in vivo by directly binding to ATP1A1 and highlight the power of our unbiased forward genetic screening approach to uncover direct drug target structures at single amino acid resolution.
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Neoplasias de la Próstata , ATPasa Intercambiadora de Sodio-Potasio , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Línea Celular Tumoral , Animales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Ratones , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacologíaRESUMEN
Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.
Asunto(s)
Apolipoproteínas E , Virus de la Fiebre Hemorrágica de Crimea-Congo , Fiebre Hemorrágica de Crimea , Receptores de LDL , Internalización del Virus , Animales , Humanos , Ratones , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Fiebre Hemorrágica de Crimea/virología , Fiebre Hemorrágica de Crimea/metabolismo , Ratones Noqueados , Receptores de LDL/metabolismo , Receptores de LDL/genética , Receptores Virales/metabolismo , Garrapatas/virología , Garrapatas/metabolismoRESUMEN
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.
Asunto(s)
Cohesinas , Proteínas Nucleares , Proteínas del Grupo Polycomb , Animales , Ratones , Cromatina/genética , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 2/genética , Proteínas del Grupo Polycomb/genética , Proteínas Nucleares/genéticaRESUMEN
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.
Asunto(s)
Células Madre Embrionarias de Ratones , Células-Madre Neurales , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular/genética , Neuronas , Factores de TranscripciónRESUMEN
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.
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Heterocromatina , N-Metiltransferasa de Histona-Lisina , Animales , Ratones , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Cromatina , Células Madre Embrionarias , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas del Tejido Nervioso/genéticaRESUMEN
Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Enfermedad del Virus de Marburg , Marburgvirus , Proteínas de Transporte Vesicular , Animales , Humanos , Ebolavirus/metabolismo , Lisosomas , Enfermedad del Virus de Marburg/genética , Enfermedad del Virus de Marburg/metabolismo , Marburgvirus/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.
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Edición Génica/métodos , Células Madre Embrionarias de Ratones/metabolismo , ARN Guía de Kinetoplastida/genética , Animales , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética , Recombinación Homóloga , Integrasas , Ratones , Mutagénesis , ARN Polimerasa IIIRESUMEN
High-density lipoproteins (HDLs) can inhibit inflammatory cytokine expression on innate immune cells, but sometimes they promote cytokine production as suggested in a recent article in Cell Metabolism by van der Vorst et al. (2017). Kopecky et al. point out that the origin, handling, and storage conditions of HDL preparations dictate their functional properties and can specifically affect immune cells to evoke a pro-inflammatory response.
Asunto(s)
Inmunidad Innata , Lipoproteínas HDL/inmunología , Animales , Citocinas/inmunología , Humanos , Inmunidad Celular , Inflamación/inmunología , Macrófagos/inmunologíaRESUMEN
Visible-light-induced antibacterial activity of carbon-doped anatase-brookite titania nano-heterojunction photocatalysts are reported for the first time. These heterostructures were prepared using a novel low temperature (100 °C) nonhydrothermal low power microwave (300 W) assisted method. Formation of interband C 2p states was found to be responsible for the band gap narrowing of the carbon doped heterojunctions. The most active photocatalyst obtained after 60 min of microwave irradiation exhibits a 2-fold higher visible-light induced photocatalytic activity in contrast to the standard commercial photocatalyst Evonik-Degussa P-25. Staphylococcus aureus inactivation rate constant for carbon-doped nano-heterojunctions and the standard photocatalyst was 0.0023 and -0.0081 min(-1), respectively. It is proposed that the photoexcited electrons (from the C 2p level) are effectively transferred from the conduction band of brookite to that of anatase causing efficient electron-hole separation, which is found to be responsible for the superior visible-light induced photocatalytic and antibacterial activities of carbon-doped anatase-brookite nano-heterojunctions.