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Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

Akute myeloische Leukämie (AML) ist eine Form der Leukämie, die aus myeloischen Vorläuferzellen entsteht und eine große zytogenetische Heterogenität sowie eine enorme molekulare Heterogenität aufweist. Charakterisiert wird sie durch unkontrollierte Proliferation und Invasion von leukämischen Zellen in umliegendes Gewebe. Leukämien entstehen aufgrund von Mutationen während der Hämatopoese, die zu abnormalem Verhalten von Zellen führen. Der Transkriptionsfaktor GATA2 ist ein wichtiger Regulator der Differenzierung von hämatopoetischen Vorläuferzellen, und deregulierte GATA2 Level wurden mit schlechten Prognosen und schweren Krankheitsverläufen assoziiert. Da die Deregulierung von GATA2 zu ungünstigen Krankheitsverläufen führt, ist die Suche nach Regulatoren von GATA2 essenziell, um eine zusätzliche therapeutische Interventionsmöglichkeit bei Patienten mit AML mit deregulierter GATA2-Expression anbieten zu können. In dieser Arbeit haben wir uns auf die Entwicklung und Validierung einer neuartigen GATA2-Reporter-Zelllinie konzentriert, die in einem genomweiten CRISPR/Cas9-Screening nach GATA2-Regulatoren eingesetzt werden kann. Dazu haben wir eine GFP Sequenz an das C-terminalen Ende des GATA2 Gens integriert und das entstandene Fusionsprotein sorgfältig validiert. Der Vergleich von Zellen, die das Fusionsprotein exprimierten mit unveränderten Zellen hat gezeigt, dass die Konstruktion des Fusionsproteins die normale GATA2-Homöostase nicht beeinträchtigt. Weiters haben wir einen CRISPR/Cas9-Screen durchgeführt, der auf bereits bekannte Interaktoren von GATA2 abgezielt hat, und bewiesen somit die Anwendbarkeit unseres Konzepts einer FACS-basierten Analyse der GATA2-Expression in der hergestellten Zelllinie. Wir schlussfolgern, dass die an GATA2 gekoppelte GFP-Analyse eines genomweiten CRISPR/Cas9-Screens praktikabel ist und dass die von uns etablierte Zelllinie dafür geeignet ist, neue Regulatoren von GATA2 zu entdecken. Wir würden vorschlagen, ein induzierbares Cas9-System zu entwickeln und einen genomweiten CRISPR/Cas9-Screen auf der Suche nach Regulatoren von GATA2 durchzuführen. Acute myeloid leukemia (AML) is a subtype of leukemia which originates from myeloid precursors and exhibits extensive cytogenetic and molecular heterogeneity. It is defined by unrestrained clonal expansion and tissue invasion of leukemic cells. The transcription factor GATA binding protein 2 (GATA2) has been shown to be a master regulator of differentiation of hematopoietic progenitors and deregulated GATA2 levels have been associated with poor prognosis and overall survival. Since deregulation of GATA2 causes unfavorable outcomes, finding upstream regulators of GATA2 is crucial for providing an additional therapeutic window in patients suffering from AML with inappropriate GATA2 expression. In this thesis, we focused on creating and validating a novel GATA2 reporter cell line, which can be used for a genome-wide CRISPR/Cas9 screen for GATA2 regulators. We tagged the GATA2 gene at the C-terminal end with a GFP sequence in the K562 leukemia cell line and carefully validated the generated fusion protein. We demonstrated that C-terminal tagging does not interfere with normal GATA2 homeostasis. We performed a focused CRISPR/Cas9 screen targeting known GATA2 interactors and proved the applicability of the model for further screens. We conclude that a genome-wide CRISPR/Cas9 screen in the K562 reporter model is feasible and will shed light on unknown GATA2 regulators. As an outlook, we propose creating an inducible system to perform a genome-wide CRISPR/Cas9 screen for GATA2 regulators. Abweichender Titel laut Übersetzung der Verfasserin/des Verfassers Masterarbeit Wien, FH Campus Wien 2023

Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus's life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.

Background Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. Methods We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. Results One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. Conclusion Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP’s surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Significance Feedback control of cholesterol metabolism is essential for cell viability and prevention of heart attacks. Cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein, which delivers cholesterol to lysosomes. To exert feedback control, cholesterol must reach the endoplasmic reticulum (ER). Here we use a CRISPR screen to show that lysosome-derived cholesterol moves first to the plasma membrane and then to the ER. The last movement requires an enzyme that produces phosphatidylserine. This demonstrates that transmembrane movement of one lipid (cholesterol) requires another lipid (phosphatidylserine). Our results explain how one organelle (ER) monitors the cholesterol content of another organelle (plasma membrane), thereby maintaining membrane integrity and ensuring cell survival., Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid—PS—controls the movement of another lipid—cholesterol—between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.

Aurora kinases are a family of serine/threonine kinases vital for cell division. Because of the overexpression of Aurora kinases in a broad range of cancers and their important roles in mitosis, inhibitors targeting Aurora kinases have attracted attention in cancer therapy. VX-680 is an effective pan-Aurora kinase inhibitor; however, its clinical efficacy was not satisfying. In this study, we performed CRISPR/Cas9 screens to identify genes whose depletion shows synthetic lethality with VX-680. The top hit from these screens was GSG2 (also known as Haspin), a serine/threonine kinase that phosphorylates histone H3 at Thr-3 during mitosis. Moreover, both Haspin knockout and Haspin inhibitor-treated HCT116 cells were hypersensitive to VX-680. Furthermore, we showed that the synthetic lethal interaction between Haspin depletion and VX-680 was mediated by the inhibition of Haspin with Aurora kinase B (AURKB), but not with Aurora kinase A (AURKA). Strikingly, combined inhibition of Haspin and AURKB had a better efficacy than single-agent treatment in both head and neck squamous cell carcinoma and non-small cell lung cancer. Taken together, our findings have uncovered a synthetic lethal interaction between AURKB and Haspin, which provides a strong rationale for this combination therapy for cancer patients.

The imipridones ONC201 and ONC212 selectively kill cancer cells and have been ascribed multiple mechanisms-of-action. Genome-wide CRISPR knockout screens revealed that loss of the mitochondrial proteases CLPP and MIPEP confer strong resistance to both compounds..., Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

Genome-wide pooled CRISPR-Cas-mediated knockout, activation, and repression screens are powerful tools for functional genomic investigations. Despite their increasing importance, there is currently little guidance on how to design and analyze CRISPR-pooled screens. Here, we provide a review of the commonly used algorithms in the computational analysis of pooled CRISPR screens. We develop a comprehensive simulation framework to benchmark and compare the performance of these algorithms using both synthetic and real datasets. Our findings inform parameter choices of CRISPR screens and provide guidance to researchers on the design and analysis of pooled CRISPR screens.

BRAF is a serine-threonine kinase that harbors activating mutations in ∼7% of human malignancies and ∼60% of melanomas. Despite initial clinical responses to BRAF inhibitors (BRAFi), patients frequently develop drug resistance. To identify candidate therapeutic targets for BRAFi-resistant melanoma, we conducted CRISPR screens in melanoma cells harboring an activating BRAF mutation that had also acquired resistance to BRAFi. The screens identified pathways and genes critical for BRAFi resistance in melanoma cells. To investigate the mechanisms and pathways enabling resistance to BRAFi in melanomas, we integrated expression data, ATAC-seq, and CRISPR screen results. We identified the JUN family of transcription factors and the ETS family transcription factor ETV5 as key regulators of CDK6 that enabled resistance to BRAFi in melanoma cells. Our findings reveal genes whose loss of function conferred resistance to a selective BRAF inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in melanoma.

Mitochondria are biosynthetic, bioenergetic, and signaling organelles with a critical role in cellular physiology. Dysfunctional mitochondria are associated with aging and underlie the cause of a wide range of diseases, from neurodegeneration to cancer. Through signaling, mitochondria regulate diverse biological outcomes. The maintenance of the mitochondrial membrane potential, for instance, is essential for proliferation, the release of mitochondrial reactive oxygen species, and oxygen sensing. The loss of mitochondrial membrane potential triggers pathways to clear damaged mitochondria and often results in cell death. In this study, we conducted a genome-wide positive selection CRISPR screen using a combination of mitochondrial inhibitors to uncover genes involved in sustaining a mitochondrial membrane potential, and therefore avoid cell death when the electron transport chain is impaired. Our screen identified genes involved in mitochondrial protein translation and ATP synthesis as essential for the induction of cell death when cells lose their mitochondrial membrane potential. This report intends to provide potential targets for the treatment of diseases associated with mitochondrial dysfunction.

Lineage-specific transcription factors have emerged as a promising class of essential genes in cancer. The best examples of leveraging this phenomenon in the clinic is targeting the androgen receptor and the oestrogen receptor in prostate and breast cancer respectively. Despite the success of these therapies, the mechanisms that maintain lineage fidelity in advanced cancer clones, and whether lineage factor pathways could be exploited in other cancer types remain poorly understood. Using clear cell renal cell carcinoma (ccRCC) as a model, I characterise mechanisms that underlie lineage factor dependence in cancer. Using CRISPR/Cas9 loss-of-function screening coupled with in vitro and in vivo validation I show that the loss of SMARCB1, a member of the SWI/SNF chromatin remodelling complex, confers an advantage to ccRCC cells upon inhibition of the essential renal lineage factor PAX8. SMARCB1 knockout (KO) leads to large-scale loss of a kidney-specific enhancer program, conversion to a cellular state resembling that of rhabdoid tumours, and the re-activation of proliferative pathways. Using a second CRISPR/Cas9 screen, I show that these proliferative pathways are underpinned by the acquisition of new transcriptional dependencies. These dependencies represent rare essential genes across different lineage-specific and oncogenic pathways, a principle validated in a large-scale CRISPR/Cas9 screening data set comprising hundreds of cancer cell lines. In summary, dependence on tissue-specific lineage factors in cancer can be modulated via epigenetic remodelling.

The clustered regularly interspaced palindromic repeats (CRISPR)-associated Cas system, derived from the defence mechanism components of prokaryotes, is frequently used as a target specific gene editing tool in in vitro and in vivo studies. Uses of the CRISPR-Cas system include gene deletion, gene insertion, base editing, gene expression editing, genomic locus imaging, epigenetic editing, and diagnostic analysis. Due to the strengths of the system such as flexibility, versatility and easy application, its usage areas have gradually increased. In recent years, CRISPR-Cas9 based functional genetic screen studies have been carried out to understand the roles of genes in the biological processes and cellular functions. CRISPR screens are used to identify genetic factors responsible for disease development and determine their functions, which are necessary for the survival of cells due to disease, especially cancer. CRISPR-Cas9 screens consisting of desing and amplification of the selected gRNA library, cloning in the relevant plasmid, packaging into lentiviruses and lentiviral transduction into cells, phenotypic selection, next generation DNA sequence analysis and bioinformatics analysis, has allowed functional genetic studies that can be performed with high-throughput and high-efficiency. Screening studies can be carried out for different purposes such as investigating pathogen-host interactions, revealing the molecular mechanisms of diseases, identifying novel therapeutic targets and revealing factors that play a key role in treatment resistance. In this review, after a short summary of different CRISPR-Cas systems the basic principles of genetic screening studies, their application and areas of usage will be discussed with selected examples from the current literature. © 2022 by Türkiye Klinikleri., Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, TÜBİTAK: 119Z280/1001, Finansal Kaynak Bu çalışma, TÜBİTAK Kurulu tarafından onaylanmış (Proje no: 119Z280/1001) ve TÜBİTAK araştırma fonunca desteklenmiştir.

Publisher Copyright: © 2022 We combined functional genomics and human genetics to investigate processes that affect type 1 diabetes (T1D) risk by mediating beta cell survival in response to proinflammatory cytokines. We mapped 38,931 cytokine-responsive candidate cis-regulatory elements (cCREs) in beta cells using ATAC-seq and snATAC-seq and linked them to target genes using co-accessibility and HiChIP. Using a genome-wide CRISPR screen in EndoC-βH1 cells, we identified 867 genes affecting cytokine-induced survival, and genes promoting survival and up-regulated in cytokines were enriched at T1D risk loci. Using SNP-SELEX, we identified 2,229 variants in cytokine-responsive cCREs altering transcription factor (TF) binding, and variants altering binding of TFs regulating stress, inflammation, and apoptosis were enriched for T1D risk. At the 16p13 locus, a fine-mapped T1D variant altering TF binding in a cytokine-induced cCRE interacted with SOCS1, which promoted survival in cytokine exposure. Our findings reveal processes and genes acting in beta cells during inflammation that modulate T1D risk.

Prostate cancer (PCa) is one of the common malignancies in male adults. Recent advances in omics technology, especially in next-generation sequencing, have increased the opportunity to identify genes that correlate with cancer diseases, including PCa. In addition, a genetic screen based on CRISPR/Cas9 technology has elucidated the mechanisms of cancer progression and drug resistance, which in turn has enabled the discovery of new targets as potential genes for new therapeutic targets. In the era of precision medicine, such knowledge is crucial for clinicians in their decision-making regarding patient treatment. In this review, we focus on how CRISPR screen for PCa performed to date has contributed to the identification of biologically critical and clinically relevant target genes.

Background Ovarian cancer, a highly metastatic malignancy, has benefited tremendously from advances in modern human genomics. However, the genomic variations related to the metastasis remains unclear. Methods We filtered various significant genes (n = 6722) associated with metastasis within a large-scale functional genomic CRISPR/Cas9 knock-out library including 122,756 single guide RNAs, and identified ITK (IL2 Inducible T Cell Kinase) as a potential cancer suppressor gene for ovarian cancer metastasis. Downstream bioinformatic analysis was performed for ITK using public databases. Results We found that patients in low-ITK group had poor prognosis and more distant metastasis than those in high-ITK group in TCGA and GEO databases. We also demonstrated that ITK combined with the clinical factors could accurately predict prognosis through multiple Cox regression analysis and ROC analysis. Moreover, alterations correlated with distant metastasis emereged with significantly increased expression in SAMRCD1 in low-ITK group, but CD244 and SOCS1 in high-ITK group. Integrated analysis revealed dysregulated molecular processes including predominantly oncogenic signaling pathways in low-ITK group but immune related pathways in high-ITK group, which suggested ITK might inhibit distant metastasis in ovarian cancer. Furtherly, deconvolution of the cellular composition of all samples validated the close correlation between ITK and immune related function especially for cytotoxic lymphocytes. Conclusions Together, these data provide insights into the potential role of ITK, with implications for the future development of tansformative ovarian cancer therapeutics.

Background In recent years, the application of functional genetic immuno-oncology screens has showcased the striking ability to identify potential regulators engaged in tumor-immune interactions. Although these screens have yielded substantial data, few studies have attempted to systematically aggregate and analyze them. Methods In this study, a comprehensive data collection of tumor immunity-associated functional screens was performed. Large-scale genomic data sets were exploited to conduct integrative analyses. Results We identified 105 regulator genes that could mediate resistance or sensitivity to immune cell-induced tumor elimination. Further analysis identified MON2 as a novel immune-oncology target with considerable therapeutic potential. In addition, based on the 105 genes, a signature named CTIS (CRISPR screening-based tumor-intrinsic immune score) for predicting response to immune checkpoint blockade (ICB) and several immunomodulatory agents with the potential to augment the efficacy of ICB were also determined. Conclusion Overall, our findings provide insights into immune oncology and open up novel opportunities for improving the efficacy of current immunotherapy agents.

Significance Systematically dissecting the function of a large set of cis-regulatory elements or transcription factor binding sites (cistromes) has been technically challenging. Using genome-wide CRISPR screens, we profiled over 10,000 FOXA1 and CTCF binding sites for their roles in regulating the fitness of breast and prostate cancer cells, and accordingly developed a model to predict essentiality for cis-elements. These efforts not only reveal how the key transcription factors and their cistromes regulate cell essentiality in hormone-dependent cancers but also highlight an efficient approach to investigate the functions of noncoding regions of the genome., Although millions of transcription factor binding sites, or cistromes, have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR/Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of over 10,000 FOXA1 and CTCF binding sites in breast and prostate cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed 2 classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at topologically associated domain (TAD) boundaries and display distinct characteristics. Using regression methods trained on our screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts noncoding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given factor and can inform the development of predictive models of cistrome function.

SUMMARY CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets., In Brief CD40 is critical for B cell development, germinal center formation, somatic hypermutation, and class-switch recombination. Increased CD40 abundance is associated with autoimmunity and cancer, whereas CD40 hypoactivity causes immunodeficiency. Jiang et al. performed a genome-wide CRISPR/Cas9 screen to reveal key B cell factors that control CD40 abundance and that regulate CD40 responses., Graphical Abstract

Significance Identifying cell-surface receptors required for viral infection is important for developing antiviral therapies and effective vaccines. Human cytomegalovirus (HCMV) infects epithelial cells during interhost transmission. The pentameric protein complex (PC) was identified as being required for infection of this clinically relevant cell type. The majority of neutralizing antibody responses are directed against the PC. Here we describe an HCMV receptor, OR14I1, which binds to the PC and defines epithelial tropism. Importantly, we identified a peptide representing the N terminus of OR14I1 that blocks HCMV infection of epithelial cells. Moreover, an adenylate cyclase signaling pathway functioning downstream of OR14I1 contributes to HCMV infection and endocytosis. These findings reveal information useful for the development of new anti-HCMV therapies and vaccines., A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH–gL–UL128–UL130–UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT–mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

Background Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. Methods We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. Results The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. Conclusions Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC. Electronic supplementary material The online version of this article (10.1186/s12885-019-5455-1) contains supplementary material, which is available to authorized users.

Formaldehyde (FA), a ubiquitous environmental pollutant, is classified as a Group I human carcinogen by the International Agency for Research on Cancer. Previously, we reported that FA induced hematotoxicity and chromosomal aneuploidy in exposed workers and toxicity in bone marrow and hematopoietic stem cells of experimental animals. Using functional toxicogenomic profiling in yeast, we identified genes and cellular processes modulating eukaryotic FA cytotoxicity. Although we validated some of these findings in yeast, many specific genes, pathways and mechanisms of action of FA in human cells are not known. In the current study, we applied genome-wide, loss-of-function CRISPR screening to identify modulators of FA toxicity in the human hematopoietic K562 cell line. We assessed the cellular genetic determinants of susceptibility and resistance to FA at 40, 100 and 150 μM (IC10, IC20 and IC60, respectively) at two time points, day 8 and day 20. We identified multiple candidate genes that increase sensitivity (e.g. ADH5, ESD and FANC family) or resistance (e.g. FASN and KDM6A) to FA when disrupted. Pathway analysis revealed a major role for the FA metabolism and Fanconi anemia pathway in FA tolerance, consistent with findings from previous studies. Additional network analyses revealed potential new roles for one-carbon metabolism, fatty acid synthesis and mTOR signaling in modulating FA toxicity. Validation of these novel findings will further enhance our understanding of FA toxicity in human cells. Our findings support the utility of CRISPR-based functional genomics screening of environmental chemicals.

Hepatitis A virus (HAV) is a positive-sense RNA virus causing acute inflammation of the liver. Here, using a genome-scale CRISPR screen in a human hepatocyte cell line, we provide a comprehensive picture of the cellular factors, which are exploited by HAV during replication. We identified genes involved in sialic acid biosynthesis and members of the eukaryotic translation initiation factor complex, corroborating their putative roles in HAV infection. Additionally, we uncovered all components of the cellular machinery for UFMylation, a ubiquitin-like protein modification. We showed that HAV translation specifically depends on UFM1 conjugation of the ribosomal protein RPL26. Furthermore, we found that components related to the yeast Trf4/5– Air1/2–Mtr4 polyadenylation (TRAMP) complex, are required for viral translation, independent of controlling HAV poly(A) tails. While the identified HAV host factors were largely distinct compared to other picornaviruses, we highlighted a surprising co-dependency of HAV and hepatitis B virus (HBV) on the TRAMP-like complex. Finally, we demonstrated that pharmacological inhibition of the TRAMP-like complex decreased HAV replication in hepatocyte cells and human liver organoids, thus providing a strategy for host-directed therapy of HAV infection.

BackgroundX-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation.ResultsTo identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression.ConclusionsHere, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

Protein secretion is an essential process in cell biology. The conventional secretion pathway is well known, and involves proteins with a signal sequence being directed into the endoplasmic reticulum (ER). Once in the lumen of the ER, proteins are trafficked through the Golgi and remain separate from the cytosol throughout their journey to the outside of the cell. More recently, proteins without a signal sequence have been found outside the cell. These proteins are secreted unconventionally, and avoid the ER-Golgi route. For both pathways, some aspects remain unclear, particularly surrounding the regulation of protein secretion. Galectin-3, an unconventionally secreted protein, was used here to investigate both conventional and unconventional protein secretion. The unconventional protein secretion of galectin-3 is poorly understood. To address this, genetic manipulation of galectin-3 was used to gain insights into the mechanism of galectin-3 secretion. It was found that galectin-3 does not require unfolding or binding to cell surface glycoproteins to be secreted. Galectin-3 was also used to assess protein secretion using a genome-wide CRISPR screen. As galectin 3 binds to cell surface glycoproteins, galectin-3 on the cell surface was used to assess secretion of these glycoproteins. Hits were validated by a glycoprotein secretion assay. Using this pipeline, 93 hits were identified as involved in protein secretion, of which 51 were novel hits not previously associated with protein secretion. Many of the hits identified also resulted in a phenotype of altered Golgi morphology, and five of these were investigated further. Two novel hits that localised to the Golgi were identified, TMEM220 and GPR161. GPR161 likely mediates its effect on Golgi morphology by its interaction with golgin A5. This thesis presents new insights into both conventional and unconventional protein secretion. Insights into the mechanism for galectin-3 secretion were revealed. A novel Golgi-localised regulator of secretion, GRP161, was identified, with a mechanism for action suggested. Importantly, the list of 51 novel genes identified will serve as a useful resource for other researchers investigating protein secretion.

Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.

Simple Summary The synthetic lethality (SL) clinical success of PARP inhibitors in homologous recombinant deficient tumors has established a new concept for cancer treatment. For decades, efforts have centered on identifying genetic interactions for determining essential tumoral genes, and more recently, SL interactions to determine combinational treatments against persistent cancer reappearance. Currently, the feasibility of CRISPR screen methodology has emerged as the state of the art for uncovering new SL or viable interactors in the biology and treatment of cancers. We present the up-to-date research of numerous laboratories that take advantage of the genome-wide forward genetic CRISPR screen tools and protocols to identify cancer biomarkers, genetic interactions and novel therapies. Indeed, investigations are nowadays focused on defining innovative combinatorial treatments based on SL interactions. By coupling different drugs, concentration treatments can be lowered and therefore toxicity reduced. CRISPR screen technologies have deeply impacted cancer research to promote a robust advance in combined therapies. Cancer is a complex disease resulting from the accumulation of genetic dysfunctions. Tumor heterogeneity causes the molecular variety that divergently controls responses to chemotherapy, leading to the recurrent problem of cancer reappearance. For many decades, efforts have focused on identifying essential tumoral genes and cancer driver mutations. More recently, prompted by the clinical success of the synthetic lethality (SL)-based therapy of the PARP inhibitors in homologous recombinant deficient tumors, scientists have centered their novel research on SL interactions (SLI). The state of the art to find new genetic interactions are currently large-scale forward genetic CRISPR screens. CRISPR technology has rapidly evolved to be a common tool in the vast majority of laboratories, as tools to implement CRISPR screen protocols are available to all researchers. Taking advantage of SLI, combinatorial therapies have become the ultimate model to treat cancer with lower toxicity, and therefore better efficiency. This review explores the CRISPR screen methodology, integrates the up-to-date published findings on CRISPR screens in the cancer field and proposes future directions to uncover cancer regulation and individual responses to chemotherapy.

The BioGRID (Biological General Repository for Interaction Datasets, thebiogrid.org) is an open‐access database resource that houses manually curated protein and genetic interactions from multiple species including yeast, worm, fly, mouse, and human. The ~1.93 million curated interactions in BioGRID can be used to build complex networks to facilitate biomedical discoveries, particularly as related to human health and disease. All BioGRID content is curated from primary experimental evidence in the biomedical literature, and includes both focused low‐throughput studies and large high‐throughput datasets. BioGRID also captures protein post‐translational modifications and protein or gene interactions with bioactive small molecules including many known drugs. A built‐in network visualization tool combines all annotations and allows users to generate network graphs of protein, genetic and chemical interactions. In addition to general curation across species, BioGRID undertakes themed curation projects in specific aspects of cellular regulation, for example the ubiquitin‐proteasome system, as well as specific disease areas, such as for the SARS‐CoV‐2 virus that causes COVID‐19 severe acute respiratory syndrome. A recent extension of BioGRID, named the Open Repository of CRISPR Screens (ORCS, orcs.thebiogrid.org), captures single mutant phenotypes and genetic interactions from published high throughput genome‐wide CRISPR/Cas9‐based genetic screens. BioGRID‐ORCS contains datasets for over 1,042 CRISPR screens carried out to date in human, mouse and fly cell lines. The biomedical research community can freely access all BioGRID data through the web interface, standardized file downloads, or via model organism databases and partner meta‐databases.

Tumor cells subvert immune surveillance by harnessing signals from immune checkpoints to acquire immune resistance. The protein PD‐L1 is an important component in this process, and inhibition of PD‐L1 elicits durable anti‐tumor responses in a broad spectrum of cancers. However, immune checkpoint inhibition that target known pathways is not universally effective. A better understanding of the genetic repertoire underlying these processes is necessary to expand our knowledge in tumor immunity and to facilitate identification of alternative targets. Here, we present a CRISPR/Cas9 screen in human cancer cells to identify genes that confer tumors with the ability to evade the cytotoxic effects of the immune system. We show that the transcriptional regulator MLLT6 (AF17) is required for efficient PD‐L1 protein expression and cell surface presentation in cancer cells. MLLT6 depletion alleviates suppression of CD8+ cytotoxic T cell‐mediated cytolysis. Furthermore, cancer cells lacking MLLT6 exhibit impaired STAT1 signaling and are insensitive to interferon‐γ‐induced stimulation of IDO1, GBP5, CD74, and MHC class II genes. Collectively, our findings establish MLLT6 as a regulator of oncogenic and interferon‐γ‐associated immune resistance., Cancer cells subvert immune surveillance by harnessing signals from immune checkpoints. MLLT6 is a modulator of the checkpoint protein PD‐L1, and its inhibition reduces immune checkpoint activity and alleviates suppression of CD8+ T cells.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs., Graphical Abstract, Highlights • Developed monkey CRISPR library to screen pathogenic coronaviruses in Vero-E6 cells • Screens identified genes that are SARS-CoV-2, MERS-CoV, and pan-coronavirus specific • Therapeutic targets, including SMARCA4, identified for SARS-CoV-2 infection • HMGB1 is novel regulator of ACE2 expression and critical for viral entry, To identify potential therapeutic targets for SARS-CoV-2 and related pathogenic coronaviruses, Wei et al. conduct genome-wide CRISPR screens in Vero-E6 cells using SARS-CoV-2, MERS-CoV, and pseudoviruses presenting SARS-CoV-1 or SARS-CoV-2 spike proteins. They identify pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively, and demonstrate that HMGB1 is critical for SARS lineage viral entry because it has a critical role in ACE2 expression.

New therapeutic targets for oral squamous cell carcinoma (OSCC) are urgently needed. We conducted genome-wide CRISPR-Cas9 screens in 21 OSCC cell lines, primarily derived from Asians, to identify genetic vulnerabilities that can be explored as therapeutic targets. We identify known and novel fitness genes and demonstrate that many previously identified OSCC-related cancer genes are non-essential and could have limited therapeutic value, while other fitness genes warrant further investigation for their potential as therapeutic targets. We validate a distinctive dependency on YAP1 and WWTR1 of the Hippo pathway, where the lost-of-fitness effect of one paralog can be compensated only in a subset of lines. We also discover that OSCCs with WWTR1 dependency signature are significantly associated with biomarkers of favorable response toward immunotherapy. In summary, we have delineated the genetic vulnerabilities of OSCC, enabling the prioritization of therapeutic targets for further exploration, including the targeting of YAP1 and WWTR1., eLife digest Many types of cancer now have 'targeted treatments', which specifically home in on genes cancer cells rely on for survival. But there are very few of these treatments available for the most common type of mouth cancer, oral squamous cell carcinoma, which around 350,000 people are diagnosed with each year. Designing targeted treatments relies on detailed knowledge of the genetic makeup of the cancer cells. But, little is known about which genes drive oral squamous cell carcinoma, especially among patients living in Asia, which is where over half of yearly cases are diagnosed. One way to resolve this is to use gene editing technology to find the genes that the cancer cells need to survive. Now, Chai et al. have used a gene editing tool known as CRISPR to examine 21 cell lines from patients diagnosed with oral squamous cell carcinoma. Most of these lines were from Asian patients, some of whom had a history of chewing betel quid which increases the risk of mouth cancer. By individually inactivating genes in these cell lines one by one, Chai et al. were able to identify 918 genes linked to the survival of the cancer cells. Some of these genes have already been associated with the spread of other types of cancer, whereas others are completely unique to oral squamous cell carcinoma. The screen also discovered that some cell lines could not survive without genes involved in a signalling pathway called Hippo, which is known to contribute to the progression of many other types of cancer. Uncovering the genes associated with oral squamous cell carcinoma opens the way for the development of new targeted treatments. Targeted therapies already exist for some of the genes identified in this study, and it may be possible to repurpose them as a treatment for this widespread mouth cancer. But, given that different cell lines relied on different genes to survive, the next step will be to identify which genes to inactivate in each patient.

The adenosine analogue remdesivir has emerged as a frontline antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness1. Prior clinical studies have identified adverse events1,2, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments7, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

Zika virus (ZIKV) is an arthropod-borne emerging pathogen causing febrile illness. ZIKV is associated with the Guillain–Barré syndrome and other neurological complications. The vertical transmission of ZIKV can cause fetus demise, stillbirths or severe congenital abnormalities and neurological complications. There is still no vaccine or specific treatment for ZIKV infection. To identify the host factors that can rescue cells from ZIKV infection, we used a genome-scale CRISPR activation screen. Our highly ranking hits included a short list of interferon-stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted interferon lambda 2 (IFN-lamda2) and interferon alpha-inducible protein 6 (IFI6) as genes providing high levels of protection from ZIKV infection. The activation of these genes had an effect at an early stage in the viral infection. In addition, infected cells expressing single guide RNAs (sgRNAs) for both of these genes displayed lower levels of cell death than did the controls. Furthermore, the identified genes were significantly induced in ZIKV-infected placenta explants. These results highlighted a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death, thus substantiating CRISPR activation screens as a valid tool for identifying host factors impeding pathogen infection.

cis-regulatory elements (CREs) regulate the expression of genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway. National Medical Research Council (NMRC) National Research Foundation (NRF) Published version We thank Yu Tao, Fang Haitong, Nickolas Teo, Aloysi Aloysius Jun-Hui Quek, and Samantha Seah for technical assistance and editorial suggestions. Y.-H.L. is supported by the NRF Investigatorship Award ( NRF12018-02 ), a JCO Development Programme Grant ( 1534n00153 ), and the National Medical Research Council ( NMRC/CBRG/0092/2015 ).

SUMMARY Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3′ UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies., Graphical Abstract, In Brief Covarrubias et al. screen ~21,000 targets, generating a resource guide of genes required for macrophage viability as well as previously unidentified positive and negative regulators of NF-κB signaling. They identify regulatory elements within essential genes and show that membrane-bound TNF primarily functions in macrophages in an autocrine fashion to negatively regulate inflammation.

Rationale: Paclitaxel resistance is a major concern when treating triple-negative breast cancer (TNBC) patients. We aimed to identify candidates causing paclitaxel resistance and explore their significance in TNBC therapeutics. Methods: A genome-wide CRISPR screening, integrated with transcriptome analyses, was performed to identify candidates involved in paclitaxel-resistant TNBCs. Cell proliferation, cytotoxicity, immunofluorescent staining, and xenograft assays were conducted to verify the phenotypes of paclitaxel resistance induced by candidate genes, both in vitro and in vivo. RNA sequencing, Western blotting, and chromatin immunoprecipitation assays were used to explore the underlying mechanisms. Results: MEF2-interacting transcriptional repressor (MITR), the truncated isoform of histone deacetylase 9 (HDAC9) lacking the deacetylation domain, was enriched in paclitaxel-resistant cells. Elevated MITR expression resulted in increased interleukin-11 (IL11) expression and activation of downstream JAK/STAT3 signaling. Mechanistically, MITR counteracted MEF2A-induced transcriptional suppression of IL11, ultimately causing paclitaxel resistance. By contrast, pharmacological inhibition of JAK1/2 by ruxolitinib reversed paclitaxel resistance both in vitro and in vivo. Conclusion: Our in vitro and in vivo genetic and cellular analyses elucidated the pivotal role of MITR/MEF2A/IL11 axis in paclitaxel resistance and provided a novel therapeutic strategy for TNBC patients to overcome poor chemotherapy responses.

Background Genome-scale pooled CRISPR screens are powerful tools for identifying genetic dependencies across varied cellular processes. The vast majority of CRISPR screens reported to date have focused exclusively on the perturbation of protein-coding gene function. However, protein-coding genes comprise Results Here, we describe a CRISPR-based screening approach that facilitates the functional profiling of thousands of noncoding regulatory elements in parallel. We selected the tumor suppressor p53 as a model system and designed a pooled CRISPR library targeting thousands of p53 binding sites throughout the genome. Following transduction into dCas9-KRAB-expressing cells we identified several regulatory elements that influence cell proliferation. Moreover, we uncovered multiple elements that are required for the p53-mediated DNA damage response. Surprisingly, many of these elements are located deep within intergenic regions of the genome that have no prior functional annotations. Conclusions This work diversifies the applications for pooled CRISPR screens and provides a framework for future functional studies focused on noncoding regulatory elements.

Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens. We discuss key issues in the analysis and design of screens, particularly the number of guides needed for faithful full discovery. Electronic supplementary material The online version of this article (10.1186/s13059-018-1538-6) contains supplementary material, which is available to authorized users.

Summary The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII., Graphical Abstract, Highlights • TMEM127 and WWP2 are required for MHCII depletion by Salmonella SteD • Tumor suppressor TMEM127 is an adaptor for oncoprotein E3 ligase WWP2 • SteD interacts with TMEM127 to induce ubiquitination and degradation of mature MHCII • SteD also undergoes TMEM127- and WWP2-dependent ubiquitination, Salmonella inhibits the adaptive immune response by reducing cell surface levels of mature MHC class II. Alix et al. reveal that the Salmonella effector SteD co-opts TMEM127, a tumor suppressor protein of previously unknown function. TMEM127 binds the E3 ubiquitin ligase WWP2 enabling ubiquitination and degradation of MHCII and SteD.

The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (i.e. three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses., eLife digest Cancer is caused by cells growing and dividing uncontrollably as a result of mutations in certain genes. Many human lung cancers have a mutation in the gene that makes the protein EGFR. In healthy cells, EGFR allows a cell to respond to chemical signals that encourage healthy growth. In cancer, the altered EGFR is always on, which allows the cell to rapidly grow without any control, resulting in cancer. One approach to treating these cancers is with drugs that block the activity of mutant EGFR. Although these drugs have been very successful, they do not always succeed in completely treating the cancer. This is because over time the cancer cells can become resistant to the drug and start forming new tumors. One way that this can happen is if random mutations lead to changes in other proteins that make the drug less effective or stop it from accessing the EGFR proteins. However, it is unclear how other proteins in cancer cells affect the response to these EGFR inhibiting drugs. Now, Zeng et al. have used gene editing to systematically remove every protein from human lung cancer cells grown in the laboratory to see how this affects resistance to EGFR inhibitor treatment. This revealed that a number of different proteins could change how cancer cells responded to the drug. For instance, cells lacking the protein RIC8A were more sensitive to EGFR inhibitors and less likely to develop resistance. This is because loss of RIC8A turns down a key cell survival pathway in cancer cells. Whereas, cancer cells lacking the ARIH2 protein were able to produce more proteins that are needed for cancer cell growth, which resulted in them having increased resistance to EGFR inhibitors. The proteins identified in this study could be used to develop new drugs that improve the effectiveness of EGFR inhibitors. Understanding how cancer cells respond to EGFR inhibitor treatment could help determine how likely a patient is to develop resistance to these drugs.

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factorsthat regulate killing by L.pneumophila. The screens reveal known host factors hijacked by L.pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L.pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators ofphagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L.pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L.pneumophila pathogenesis and mammalian cell function.

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.

As our understanding of the cancer genome has progressed, traditional chemotherapeutic agents are being replaced, in part, by targeted therapies. Development of these therapies is driven by our understanding of genetic vulnerabilities harboured by tumours. The aim of this project is to identify novel synthetic lethal interactions within melanoma. Synthetic lethality describes a relationship between two genes, where loss of either of the pair is compatible with cellular viability, but simultaneous loss of both genes induces cell death. This approach can therefore exploit somatically acquired mutations to specifically kill tumour cells and to define new therapeutic targets. Abstract In order to do this, we selected a panel of putative synthetic lethal interactions and built a bespoke multiplex CRISPR-Cas9 library to interrogate 1192 gene-pair interactions. We deployed this library on a panel of melanoma cell lines and a retinal pigment epithelial cell line as a normal comparator. We went on to develop a bioinformatic pipeline with which to analyse the paired screen output and using this pipeline we have identified a number of novel synthetic lethal relationships. Using low throughput assays we validated a number of the interactions found by our screen. Finally, using human tumour expression data, we identified a candidate pair with potential therapeutic relevance. Using external datasets and isogenic knockout models we have further validated this relationship and performed mechanistic experiments to explore the role of these genes within the cell and generate a hypothesis to explain why loss of these two genes results in cell death., Sanger Wellcome Trust Clinician Fellowship

Summary Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we chose Sall1 for further investigation. We show that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate that Sall1 synergizes with Nanog in reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validate Klf5 and Fam189a2 as new downstream targets of Sall1 and Nanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming., Graphical Abstract, Highlights • Genome-scale CRISPRa screen in mouse EpiSCs identifies novel reprogramming factors • 50 novel genes, including Sall1 and Fam189a2, identified to mediate reprogramming • Sall1 synergizes with Nanog to increase reprogramming efficiency in EpiSCs and MEFs • RNA-seq provides insight into downstream pathways of Sall1 and Nanog-mediated reprogramming, In this study, Metzakopian and colleagues use CRISPR activation to perform a genome-scale reprogramming screen on mouse epiblast stem cells identifying 50 novel candidates. In addition, the authors provide evidence of Sall1 being a potent reprogramming gene, capable of synergizing with Nanog. Lastly, using RNA-seq, the authors provide insight into potential downstream targets of Sall1 and Nanog.

Simple Summary The synthetic lethality (SL) clinical success of PARP inhibitors in homologous recombinant deficient tumors has established a new concept for cancer treatment. For decades, efforts have centered on identifying genetic interactions for determining essential tumoral genes, and more recently, SL interactions to determine combinational treatments against persistent cancer reappearance. Currently, the feasibility of CRISPR screen methodology has emerged as the state of the art for uncovering new SL or viable interactors in the biology and treatment of cancers. We present the up-to-date research of numerous laboratories that take advantage of the genome-wide forward genetic CRISPR screen tools and protocols to identify cancer biomarkers, genetic interactions and novel therapies. Indeed, investigations are nowadays focused on defining innovative combinatorial treatments based on SL interactions. By coupling different drugs, concentration treatments can be lowered and therefore toxicity reduced. CRISPR screen technologies have deeply impacted cancer research to promote a robust advance in combined therapies. Abstract Cancer is a complex disease resulting from the accumulation of genetic dysfunctions. Tumor heterogeneity causes the molecular variety that divergently controls responses to chemotherapy, leading to the recurrent problem of cancer reappearance. For many decades, efforts have focused on identifying essential tumoral genes and cancer driver mutations. More recently, prompted by the clinical success of the synthetic lethality (SL)-based therapy of the PARP inhibitors in homologous recombinant deficient tumors, scientists have centered their novel research on SL interactions (SLI). The state of the art to find new genetic interactions are currently large-scale forward genetic CRISPR screens. CRISPR technology has rapidly evolved to be a common tool in the vast majority of laboratories, as tools to implement CRISPR screen protocols are available to all researchers. Taking advantage of SLI, combinatorial therapies have become the ultimate model to treat cancer with lower toxicity, and therefore better efficiency. This review explores the CRISPR screen methodology, integrates the up-to-date published findings on CRISPR screens in the cancer field and proposes future directions to uncover cancer regulation and individual responses to chemotherapy.

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens invivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.

Summary To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer., Graphical Abstract, Highlights • Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI • Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges • Distinct ligand-receptor and spatial niche associations for tumor and stromal cells. • Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening, Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.