RESUMEN
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
Asunto(s)
Sistemas CRISPR-Cas , Reprogramación Celular , Epigénesis Genética , Epigenoma , Edición Génica , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Diferenciación Celular , Islas de CpG , Metilación de ADN , Silenciador del Gen , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.
Asunto(s)
Biomarcadores/metabolismo , Colesterol/metabolismo , Epistasis Genética , Redes Reguladoras de Genes , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Jurkat , Células K562 , Mapeo de Interacción de ProteínasRESUMEN
While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.
Asunto(s)
Variación Estructural del Genoma/genética , Neoplasias de la Próstata/genética , Anciano , Anciano de 80 o más Años , Proteína BRCA2/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Variaciones en el Número de Copia de ADN , Exoma , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Secuencias Repetidas en Tándem/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuenciación Completa del Genoma/métodosRESUMEN
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting â¼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endorribonucleasas , Retroalimentación , Humanos , Modelos Moleculares , Proteínas Serina-Treonina Quinasas , ARN Guía de Kinetoplastida/metabolismo , Transcripción Genética , Respuesta de Proteína DesplegadaRESUMEN
Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthetic multigene transcriptional programs in which some genes are activated and others are repressed. We apply this approach to flexibly redirect flux through a complex branched metabolic pathway in yeast. Moreover, these programs can be executed by inducing expression of the dCas9 protein, which acts as a single master regulatory control point. CRISPR-associated RNA scaffolds provide a powerful way to construct synthetic gene expression programs for a wide range of applications, including rewiring cell fates or engineering metabolic pathways.
Asunto(s)
Sistemas CRISPR-Cas , Expresión Génica , Técnicas Genéticas , Células HEK293 , Humanos , Ingeniería Metabólica , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae/genética , Streptococcus pyogenes/genéticaRESUMEN
CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.
Asunto(s)
Alelos , Edición Génica , Mutagénesis , Linfocitos T , Humanos , Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Mutagénesis/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Activación de Linfocitos , Citocinas/biosíntesis , Citocinas/metabolismo , Mutación con Ganancia de Función , Mutación con Pérdida de FunciónRESUMEN
Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Asunto(s)
Edición Génica , Proteínas de Unión al ARN , Humanos , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Células K562 , Poli U/genética , Poli U/metabolismo , ARN Polimerasa III/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.
Asunto(s)
Imagen Molecular/métodos , Imagen Óptica/métodos , Multimerización de Proteína , Proteínas/química , Animales , Sistemas CRISPR-Cas , Técnicas Genéticas , Humanos , Anticuerpos de Cadena Única/químicaRESUMEN
While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a â¼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas Genéticas , Transcripción Genética , Línea Celular , Toxina del Cólera/metabolismo , Toxina Diftérica/metabolismo , Genoma Humano , HumanosRESUMEN
The past 25 years of genomics research first revealed which genes are encoded by the human genome and then a detailed catalogue of human genome variation associated with many diseases. Despite this, the function of many genes and gene regulatory elements remains poorly characterized, which limits our ability to apply these insights to human disease. The advent of new CRISPR functional genomics tools allows for scalable and multiplexable characterization of genes and gene regulatory elements encoded by the human genome. These approaches promise to reveal mechanisms of gene function and regulation, and to enable exploration of how genes work together to modulate complex phenotypes.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Humano/genética , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Polimorfismo de Nucleótido Simple , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/genética , Estudios de Asociación Genética/métodos , Genómica/tendencias , HumanosRESUMEN
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Asunto(s)
Endodesoxirribonucleasas/genética , Escherichia coli/genética , Técnicas de Silenciamiento del Gen/métodos , Interferencia de ARN , Streptococcus pyogenes/enzimología , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Expresión Génica , Streptococcus pyogenes/genética , Elongación de la Transcripción Genética , Iniciación de la Transcripción Genética , ARN Pequeño no TraducidoRESUMEN
The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.
Asunto(s)
Técnicas Genéticas , Telómero , Secuencia de Bases , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Mitosis , Datos de Secuencia Molecular , Mucina 4/genéticaRESUMEN
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Asunto(s)
Proteínas Bacterianas/genética , Marcación de Gen/métodos , Streptococcus pyogenes , Células HEK293 , Células HeLa , Humanos , Saccharomyces cerevisiae/genética , ARN Pequeño no TraducidoRESUMEN
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Neoplasias/patología , Preparaciones Farmacéuticas/metabolismo , Sulfonas/farmacología , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Contaminación de Medicamentos , Glicina/farmacología , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Preparaciones Farmacéuticas/química , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3' untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
RESUMEN
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.
Asunto(s)
Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Inmunidad Innata , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismoRESUMEN
While numerous cell-intrinsic processes are known to play decisive roles in chemotherapeutic response, relatively little is known about the impact of the tumor microenvironment on therapeutic outcome. Here, we use a well-established mouse model of Burkitt's lymphoma to show that paracrine factors in the tumor microenvironment modulate lymphoma cell survival following the administration of genotoxic chemotherapy. Specifically, IL-6 and Timp-1 are released in the thymus in response to DNA damage, creating a "chemo-resistant niche" that promotes the survival of a minimal residual tumor burden and serves as a reservoir for eventual tumor relapse. Notably, IL-6 is released acutely from thymic endothelial cells in a p38-dependent manner following genotoxic stress, and this acute secretory response precedes the gradual induction of senescence in tumor-associated stromal cells. Thus, conventional chemotherapies can induce tumor regression while simultaneously eliciting stress responses that protect subsets of tumor cells in select anatomical locations from drug action.
Asunto(s)
Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/patología , Daño del ADN , Resistencia a Antineoplásicos , Comunicación Paracrina , Timo/metabolismo , Animales , Linfoma de Burkitt/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular , Senescencia Celular , Medios de Cultivo Condicionados , Citocinas/análisis , Citocinas/metabolismo , Doxorrubicina/uso terapéutico , Células Endoteliales/metabolismo , Humanos , Interleucina-6/metabolismo , Janus Quinasa 2/metabolismo , Neoplasias Hepáticas/patología , Ratones , Neoplasia Residual/metabolismo , Neoplasia Residual/patología , Timo/química , Timo/citología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.