RESUMEN
Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum's adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.
Asunto(s)
Sistemas CRISPR-Cas , Hepatocitos , Malaria Falciparum , Plasmodium falciparum , Theileria , Plasmodium falciparum/genética , Humanos , Hepatocitos/parasitología , Hepatocitos/metabolismo , Malaria Falciparum/parasitología , Theileria/genética , Genómica/métodos , Hemo/metabolismo , Interacciones Huésped-Parásitos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Animales , Técnicas de Inactivación de GenesRESUMEN
Small cell lung cancers (SCLCs) are composed of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLCs, â¼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes as top dependencies specific to POU2F3-positive SCLC. Notably, chemical disruption of mSWI/SNF ATPase activity attenuates proliferation of all POU2F3-positive SCLCs, while disruption of non-canonical BAF (ncBAF) via BRD9 degradation is effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF targets to and maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, clinical-grade pharmacologic disruption of SMARCA4/2 ATPases and BRD9 decreases POU2F3-SCLC tumor growth and increases survival in vivo. These results demonstrate mSWI/SNF-mediated governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for POU2F3-positive SCLCs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones , Línea Celular Tumoral , Proliferación Celular , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genéticaRESUMEN
Reducing disparities is vital for equitable access to precision treatments in cancer. Socioenvironmental factors are a major driver of disparities, but differences in genetic variation likely also contribute. The impact of genetic ancestry on prioritization of cancer targets in drug discovery pipelines has not been systematically explored due to the absence of pre-clinical data at the appropriate scale. Here, we analyze data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models to identify ancestry-associated genetic dependencies essential for cell survival. Surprisingly, we find that most putative associations between ancestry and dependency arise from artifacts related to germline variants. Our analysis suggests that for 1.2-2.5% of guides, germline variants in sgRNA targeting sequences reduce cutting by the CRISPR/Cas9 nuclease, disproportionately affecting cell models derived from individuals of recent African descent. We propose three approaches to mitigate this experimental bias, enabling the scientific community to address these disparities.
Asunto(s)
Sistemas CRISPR-Cas , Mutación de Línea Germinal , Humanos , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Células Germinativas/metabolismo , Variación Genética , Neoplasias/genética , Reacciones Falso Negativas , Genoma Humano , Línea Celular Tumoral , Línea CelularRESUMEN
Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib , Leucemia , Transducción de Señal , Quinasas p21 Activadas , Animales , Humanos , Ratones , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Citarabina/farmacología , Citarabina/uso terapéutico , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Leucemia/genética , Leucemia/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismo , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.
Asunto(s)
Proteínas Bacterianas , Sistemas CRISPR-Cas , Endodesoxirribonucleasas , Técnicas de Inactivación de Genes , Humanos , Técnicas de Inactivación de Genes/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Biblioteca de Genes , Línea Celular Tumoral , Genes Esenciales , Células HEK293 , Epistasis Genética , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismoRESUMEN
The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodosRESUMEN
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
Asunto(s)
Procesamiento Proteico-Postraduccional , Fosforilación , Humanos , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/genética , Transducción de Señal , Células HEK293 , Proteómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Linfocitos T/metabolismo , Células Jurkat , FN-kappa B/metabolismoRESUMEN
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Asunto(s)
Sistemas CRISPR-Cas , Hexosiltransferasas , Lipopolisacáridos , Proteínas de la Membrana , FN-kappa B , Transducción de Señal , Receptor Toll-Like 4 , Hexosiltransferasas/metabolismo , Hexosiltransferasas/genética , FN-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Receptor Toll-Like 4/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Células HEK293 , Inflamación/metabolismo , Inflamación/genética , Glicosilación , Microscopía por Crioelectrón , Dominio Catalítico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Vectores Genéticos/genéticaRESUMEN
The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection.
Asunto(s)
Regulación de la Expresión Génica , Inmunidad Innata , Proteínas de la Membrana , FN-kappa B , Proteínas Oncogénicas , Proteínas de Unión al ARN , Animales , Humanos , Sistemas CRISPR-Cas , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Células HEK293 , Inmunidad Innata/genética , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Transducción de Señal , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/inmunologíaRESUMEN
In vivo T cell screens are a powerful tool for elucidating complex mechanisms of immunity, yet there is a lack of consensus on the screen design parameters required for robust in vivo screens: gene library size, cell transfer quantity, and number of mice. Here, we describe the Framework for In vivo T cell Screens (FITS) to provide experimental and analytical guidelines to determine optimal parameters for diverse in vivo contexts. As a proof-of-concept, we used FITS to optimize the parameters for a CD8+ T cell screen in the B16-OVA tumor model. We also included unique molecular identifiers (UMIs) in our screens to (1) improve statistical power and (2) track T cell clonal dynamics for distinct gene knockouts (KOs) across multiple tissues. These findings provide an experimental and analytical framework for performing in vivo screens in immune cells and illustrate a case study for in vivo T cell screens with UMIs.
Asunto(s)
Linfocitos T CD8-positivos , Animales , Ratones , Técnicas de Inactivación de GenesRESUMEN
Small cell lung cancers (SCLC) are comprised of heterogeneous subtypes marked by lineage-specific transcription factors, including ASCL1, NEUROD1, and POU2F3. POU2F3-positive SCLC, â¼12% of all cases, are uniquely dependent on POU2F3 itself; as such, approaches to attenuate POU2F3 expression may represent new therapeutic opportunities. Here using genome-scale screens for regulators of POU2F3 expression and SCLC proliferation, we define mSWI/SNF complexes, including non-canonical BAF (ncBAF) complexes, as top dependencies specific to POU2F3-positive SCLC. Notably, clinical-grade pharmacologic mSWI/SNF inhibition attenuates proliferation of all POU2F3-positive SCLCs, while disruption of ncBAF via BRD9 degradation is uniquely effective in pure non-neuroendocrine POU2F3-SCLCs. mSWI/SNF maintains accessibility over gene loci central to POU2F3-mediated gene regulatory networks. Finally, chemical targeting of SMARCA4/2 mSWI/SNF ATPases and BRD9 decrease POU2F3-SCLC tumor growth and increase survival in vivo . Taken together, these results characterize mSWI/SNF-mediated global governance of the POU2F3 oncogenic program and suggest mSWI/SNF inhibition as a therapeutic strategy for SCLC.
RESUMEN
ABSTRACT: Aberrant expression of stem cell-associated genes is a common feature in acute myeloid leukemia (AML) and is linked to leukemic self-renewal and therapy resistance. Using AF10-rearranged leukemia as a prototypical example of the recurrently activated "stemness" network in AML, we screened for chromatin regulators that sustain its expression. We deployed a CRISPR-Cas9 screen with a bespoke domain-focused library and identified several novel chromatin-modifying complexes as regulators of the TALE domain transcription factor MEIS1, a key leukemia stem cell (LSC)-associated gene. CRISPR droplet sequencing revealed that many of these MEIS1 regulators coordinately controlled the transcription of several AML oncogenes. In particular, we identified a novel role for the Tudor-domain-containing chromatin reader protein SGF29 in the transcription of AML oncogenes. Furthermore, SGF29 deletion impaired leukemogenesis in models representative of multiple AML subtypes in multiple AML subtype models. Our studies reveal a novel role for SGF29 as a nononcogenic dependency in AML and identify the SGF29 Tudor domain as an attractive target for drug discovery.
Asunto(s)
Proteínas de Homeodominio , Leucemia Mieloide Aguda , Humanos , Proteínas de Homeodominio/genética , Cromatina/genética , Factores de Transcripción/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , CarcinogénesisRESUMEN
Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
Asunto(s)
Sistemas CRISPR-Cas , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Ratones , Animales , Sistemas CRISPR-Cas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Ratones Noqueados , Sistema Inmunológico , Edición GénicaRESUMEN
To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.
Asunto(s)
Toxoplasma , Toxoplasmosis , Humanos , Interferón gamma/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Toxoplasma/metabolismo , Factores de Transcripción , Adenosina Trifosfatasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.
Asunto(s)
Linfoma de Burkitt , Leucemia , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , ARN Guía de Sistemas CRISPR-Cas , Inmunoterapia Adoptiva , Linfocitos T , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Microambiente TumoralRESUMEN
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
RESUMEN
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
RESUMEN
The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape we conducted a gain of function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including JADE3 a protein involved in directing the histone acetyltransferase HBO1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Interestingly, expression of the closely related paralogues JADE1 and JADE2 are unable to restrict influenza A virus infection, suggesting a distinct function of JADE3. We identify both shared and unique transcriptional signatures between uninfected cells expressing JADE3 and JADE2. These data provide a framework for understanding the overlapping and distinct functions of the JADE family of paralogues. Specifically, we find that JADE3 expression activates the NF-kB signaling pathway, consistent with an antiviral function. Therefore, we propose JADE3, but not JADE1 or JADE2, activates an antiviral genetic program involving the NF-kB pathway to restrict influenza A virus infection.