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1.
Cell ; 184(1): 92-105.e16, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33147445

RESUMO

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.


Assuntos
COVID-19/genética , COVID-19/virologia , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Vias Biossintéticas , COVID-19/metabolismo , Colesterol/biossíntese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endossomos/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes/métodos , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferência de RNA , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Carga Viral/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
2.
Cell ; 160(6): 1246-60, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25748654

RESUMO

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.


Assuntos
Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/genética , Técnicas de Inativação de Genes , Neoplasias Pulmonares/genética , Metástase Neoplásica/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Pulmonares/patologia , Camundongos , RNA Guia de Cinetoplastídeos
3.
Cell ; 162(3): 675-86, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26189680

RESUMO

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.


Assuntos
Sistemas CRISPR-Cas , Técnicas Genéticas , Imunidade Inata , Animais , Células da Medula Óssea/imunologia , Diferenciação Celular , Sobrevivência Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
4.
Mol Cell ; 82(2): 221-226, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35063087

RESUMO

With the focus on technology for this issue of Molecular Cell, a group of scientists working in different areas of molecular biology provide their perspective on the most recent important technological advance in their field, where the field is lacking, and their wish list for future technology development.


Assuntos
Pesquisa Biomédica/tendências , Técnicas Genéticas/tendências , Biologia Molecular/tendências , Animais , Difusão de Inovações , Humanos
6.
Nature ; 603(7902): 728-735, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35296855

RESUMO

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Linfócitos T CD4-Positivos , Proliferação de Células , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/genética
7.
Nature ; 602(7896): 268-273, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35110736

RESUMO

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.


Assuntos
Transtorno do Espectro Autista , Predisposição Genética para Doença , Neurônios , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Córtex Cerebral/citologia , Proteínas de Ligação a DNA/genética , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Histona-Lisina N-Metiltransferase/genética , Humanos , Neurônios/classificação , Neurônios/metabolismo , Neurônios/patologia , Organoides/citologia , Proteômica , RNA-Seq , Análise de Célula Única , Fatores de Transcrição/genética
8.
Trends Genet ; 40(2): 118-133, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37989654

RESUMO

Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Genoma , Testes Genéticos , Análise de Célula Única/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
9.
Proc Natl Acad Sci U S A ; 121(21): e2403685121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38743625

RESUMO

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.


Assuntos
Quinases Proteína-Quinases Ativadas por AMP , Fosfatidilinositol 3-Quinases , Proteínas Serina-Treonina Quinases , Esferoides Celulares , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Quinases Proteína-Quinases Ativadas por AMP/genética , Esferoides Celulares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proliferação de Células , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
10.
Am J Hum Genet ; 110(10): 1750-1768, 2023 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-37802044

RESUMO

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Humanos , Animais , Camundongos , Transtorno Autístico/genética , Transtorno do Espectro Autista/genética , Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Fatores de Transcrição/genética
11.
Nat Methods ; 20(1): 86-94, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36550277

RESUMO

Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.


Assuntos
Cromatina , RNA , RNA/genética , Sistemas CRISPR-Cas/genética
12.
Mol Cell ; 68(1): 44-59, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28985510

RESUMO

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Intergênico/genética , Endonucleases/genética , Edição de Genes/métodos , Genoma , Animais , DNA Intergênico/metabolismo , Endonucleases/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Engenharia Genética , Biblioteca Genômica , Ensaios de Triagem em Larga Escala , Humanos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
13.
Nature ; 548(7667): 343-346, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28792927

RESUMO

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Loci Gênicos/genética , Genoma Humano/genética , Indóis/farmacologia , Melanoma/genética , RNA Longo não Codificante/genética , Sulfonamidas/farmacologia , Ativação Transcricional/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Sítio de Iniciação de Transcrição , Vemurafenib
15.
Nature ; 543(7644): 270-274, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28241139

RESUMO

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/metabolismo , Domínios Proteicos , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Edição de Genes , Genoma/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteólise , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética
16.
Nature ; 548(7669): 537-542, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28783722

RESUMO

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.


Assuntos
Genes Essenciais/genética , Imunoterapia , Neoplasias/genética , Neoplasias/terapia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Feminino , Genoma/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/imunologia , Janus Quinase 1/metabolismo , Bases de Conhecimento , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Mutação , Neoplasias/imunologia , Neoplasias/metabolismo , Reprodutibilidade dos Testes , Linfócitos T Citotóxicos/metabolismo
17.
Nat Methods ; 16(5): 409-412, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011186

RESUMO

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Camundongos , Células NIH 3T3 , RNA Guia de Cinetoplastídeos/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas
18.
Nat Rev Genet ; 16(5): 299-311, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25854182

RESUMO

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.


Assuntos
Sistemas CRISPR-Cas , Genômica/métodos , Animais , Técnicas de Inativação de Genes , Biblioteca Gênica , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Modelos Genéticos , Interferência de RNA
19.
Nature ; 527(7577): 192-7, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26375006

RESUMO

Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Proteínas de Transporte/genética , Elementos Facilitadores Genéticos/genética , Engenharia Genética , Mutagênese/genética , Proteínas Nucleares/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA , Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Genoma/genética , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Guia de Cinetoplastídeos/genética , Proteínas Repressoras , Reprodutibilidade dos Testes , Especificidade da Espécie
20.
FASEB J ; 33(4): 5287-5299, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30698461

RESUMO

Overexpression of mouse neurogenin ( Neurog) 2 alone or in combination with mouse Neurog2/1 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can rapidly produce high-yield excitatory neurons. Here, we report a detailed characterization of human neuronal networks induced by the expression of human NEUROG2 together with human NEUROG2/1 in hESCs using molecular, cellular, and electrophysiological measurements over 60 d after induction. Both excitatory synaptic transmission and network firing activity increased over time. Strikingly, inhibitory synaptic transmission and GABAergic cells were identified from NEUROG2/1 induced neurons (iNs). To illustrate the application of such iNs, we demonstrated that the heterozygous knock out of SCN2A, whose loss-of-function mutation is strongly implicated in autism risk, led to a dramatic reduction in network activity in the NEUROG2/1 iNs. Our findings not only extend our understanding of the NEUROG2/1-induced human neuronal network but also substantiate NEUROG2/1 iNs as an in vitro system for modeling neuronal and functional deficits on a human genetic background.-Lu, C., Shi, X., Allen, A., Baez-Nieto, D., Nikish, A., Sanjana, N. E., Pan, J. Q. Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Imuno-Histoquímica , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia
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