Thijn R. Brummelkamp

Bio: Thijn R. Brummelkamp is an academic researcher from Netherlands Cancer Institute. The author has contributed to research in topics: Genetic screen & Gene. The author has an hindex of 56, co-authored 110 publications receiving 23682 citations. Previous affiliations of Thijn R. Brummelkamp include Stanford University & Austrian Academy of Sciences.

A System for Stable Expression of Short Interfering RNAs in Mammalian Cells

Abstract: Mammalian genetic approaches to study gene function have been hampered by the lack of tools to generate stable loss-of-function phenotypes efficiently. We report here a new vector system, named pSUPER, which directs the synthesis of small interfering RNAs (siRNAs) in mammalian cells. We show that siRNA expression mediated by this vector causes efficient and specific down-regulation of gene expression, resulting in functional inactivation of the targeted genes. Stable expression of siRNAs using this vector mediates persistent suppression of gene expression, allowing the analysis of loss-of-function phenotypes that develop over longer periods of time. Therefore, the pSUPER vector constitutes a new and powerful system to analyze gene function in a variety of mammalian cell types.

Regulation of progenitor cell proliferation and granulocyte function by microRNA-223

Abstract: MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes1,2. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response.

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Abstract: The extraordinary virulence of the Ebola and Marburg filoviruses has spurred intensive research into the molecular mechanisms by which they multiply and cause disease. Carette et al. use a genome-wide genetic screen in human cells to identify factors required for entry of Ebola virus. The screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cote et al. report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection in cell culture, with NPC1 being the target. The unexpected role for the hereditary disease gene NPC1 in Ebola virus infection may facilitate the development of antifilovirus therapeutics. Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3,4,5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

RNA-Guided Human Genome Engineering via Cas9

Abstract: Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

A General Framework for Weighted Gene Co-Expression Network Analysis

Abstract: Gene co-expression networks are increasingly used to explore the system-level functionality of genes. The network construction is conceptually straightforward: nodes represent genes and nodes are connected if the corresponding genes are significantly co-expressed across appropriately chosen tissue samples. In reality, it is tricky to define the connections between the nodes in such networks. An important question is whether it is biologically meaningful to encode gene co-expression using binary information (connected=1, unconnected=0). We describe a general framework for ;soft' thresholding that assigns a connection weight to each gene pair. This leads us to define the notion of a weighted gene co-expression network. For soft thresholding we propose several adjacency functions that convert the co-expression measure to a connection weight. For determining the parameters of the adjacency function, we propose a biologically motivated criterion (referred to as the scale-free topology criterion). We generalize the following important network concepts to the case of weighted networks. First, we introduce several node connectivity measures and provide empirical evidence that they can be important for predicting the biological significance of a gene. Second, we provide theoretical and empirical evidence that the ;weighted' topological overlap measure (used to define gene modules) leads to more cohesive modules than its ;unweighted' counterpart. Third, we generalize the clustering coefficient to weighted networks. Unlike the unweighted clustering coefficient, the weighted clustering coefficient is not inversely related to the connectivity. We provide a model that shows how an inverse relationship between clustering coefficient and connectivity arises from hard thresholding. We apply our methods to simulated data, a cancer microarray data set, and a yeast microarray data set.

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Abstract: Summary Background Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. Methods In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. Findings 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p Interpretation The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/mL could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.

Cancer genes and the pathways they control.

Abstract: The revolution in cancer research can be summed up in a single sentence: cancer is, in essence, a genetic disease. In the last decade, many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified, and the pathways through which they act characterized. The purposes of this review are to highlight examples of progress in these areas, indicate where knowledge is scarce and point out fertile grounds for future investigation.

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

Abstract: The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12 , as well as novel hits NF2 , CUL3 , TADA2B , and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.