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1.
Cell Rep ; 36(11): 109697, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525371

RESUMO

Midbrain dopaminergic (mDA) neurons are diverse in their projection targets, effect on behavior, and susceptibility to neurodegeneration. Little is known about the molecular mechanisms establishing this diversity during development. We show that the transcription factor BCL11A is expressed in a subset of mDA neurons in the developing and adult murine brain and in a subpopulation of pluripotent-stem-cell-derived human mDA neurons. By combining intersectional labeling and viral-mediated tracing, we demonstrate that Bcl11a-expressing mDA neurons form a highly specific subcircuit within the murine dopaminergic system. In the substantia nigra, the Bcl11a-expressing mDA subset is particularly vulnerable to neurodegeneration upon α-synuclein overexpression or oxidative stress. Inactivation of Bcl11a in murine mDA neurons increases this susceptibility further, alters the distribution of mDA neurons, and results in deficits in skilled motor behavior. In summary, BCL11A defines mDA subpopulations with highly distinctive characteristics and is required for establishing and maintaining their normal physiology.

3.
Genome Biol ; 22(1): 43, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33482885

RESUMO

BACKGROUND: Microexons, exons that are ≤ 30 nucleotides, are a highly conserved and dynamically regulated set of cassette exons. They have key roles in nervous system development and function, as evidenced by recent results demonstrating the impact of microexons on behaviour and cognition. However, microexons are often overlooked due to the difficulty of detecting them using standard RNA-seq aligners. RESULTS: Here, we present MicroExonator, a novel pipeline for reproducible de novo discovery and quantification of microexons. We process 289 RNA-seq datasets from eighteen mouse tissues corresponding to nine embryonic and postnatal stages, providing the most comprehensive survey of microexons available for mice. We detect 2984 microexons, 332 of which are differentially spliced throughout mouse embryonic brain development, including 29 that are not present in mouse transcript annotation databases. Unsupervised clustering of microexons based on their inclusion patterns segregates brain tissues by developmental time, and further analysis suggests a key function for microexons in axon growth and synapse formation. Finally, we analyse single-cell RNA-seq data from the mouse visual cortex, and for the first time, we report differential inclusion between neuronal subpopulations, suggesting that some microexons could be cell type-specific. CONCLUSIONS: MicroExonator facilitates the investigation of microexons in transcriptome studies, particularly when analysing large volumes of data. As a proof of principle, we use MicroExonator to analyse a large collection of both mouse bulk and single-cell RNA-seq datasets. The analyses enabled the discovery of previously uncharacterized microexons, and our study provides a comprehensive microexon inclusion catalogue during mouse development.

4.
Autophagy ; 17(6): 1543-1554, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32486891

RESUMO

The 21st century has revealed much about the fundamental cellular process of autophagy. Autophagy controls the catabolism and recycling of various cellular components both as a constitutive process and as a response to stress and foreign material invasion. There is considerable knowledge of the molecular mechanisms of autophagy, and this is still growing as new modalities emerge. There is a need to investigate autophagy mechanisms reliably, comprehensively and conveniently. Reactome is a freely available knowledgebase that consists of manually curated molecular events (reactions) organized into cellular pathways (https://reactome.org/). Pathways/reactions in Reactome are hierarchically structured, graphically presented and extensively annotated. Data analysis tools, such as pathway enrichment, expression data overlay and species comparison, are also available. For customized analysis, information can also be programmatically queried. Here, we discuss the curation and annotation of the molecular mechanisms of autophagy in Reactome. We also demonstrate the value that Reactome adds to research by reanalyzing a previously published work on genome-wide CRISPR screening of autophagy components.Abbreviations: CMA: chaperone-mediated autophagy; GO: Gene Ontology; MA: macroautophagy; MI: microautophagy; MTOR: mechanistic target of rapamycin kinase; SQSTM1: sequestosome 1.

5.
Cell Death Differ ; 28(6): 1804-1821, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33335290

RESUMO

Mitochondria-ER contact sites (MERCS) are known to underpin many important cellular homoeostatic functions, including mitochondrial quality control, lipid metabolism, calcium homoeostasis, the unfolded protein response and ER stress. These functions are known to be dysregulated in neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD) and amyloid lateral sclerosis (ALS), and the number of disease-related proteins and genes being associated with MERCS is increasing. However, many details regarding MERCS and their role in neurodegenerative diseases remain unknown. In this review, we aim to summarise the current knowledge regarding the structure and function of MERCS, and to update the field on current research in PD, AD and ALS. Furthermore, we will evaluate high-throughput screening techniques, including RNAi vs CRISPR/Cas9, pooled vs arrayed formats and how these could be combined with current techniques to visualise MERCS. We will consider the advantages and disadvantages of each technique and how it can be utilised to uncover novel protein pathways involved in MERCS dysfunction in neurodegenerative diseases.

6.
iScience ; 23(11): 101703, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33196026

RESUMO

Advances in single cell genomics and transcriptomics have shown that at tissue level there is complex cellular heterogeneity. To understand the effect of this inter-cell heterogeneity on metabolism it is essential to develop a single cell lipid profiling approach that allows the measurement of lipids in large numbers of single cells from a population. This will provide a functional readout of cell activity and membrane structure. Using liquid extraction surface analysis coupled with high-resolution mass spectrometry we have developed a high-throughput method for untargeted single cell lipid profiling. This technological advance highlighted the importance of cellular heterogeneity in the functional metabolism of individual human dopamine neurons, suggesting that A53T alpha-synuclein (SNCA) mutant neurons have impaired membrane function. These results demonstrate that this single cell lipid profiling platform can provide robust data that will expand the frontiers in biomedical research.

7.
Cell Rep ; 33(2): 108263, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33053338

RESUMO

The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest single-cell transcriptomic study of human iPSC-derived dopaminergic neurons to elucidate gene expression dynamics in response to cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles and differential sensitivity to stress, highlighting cellular heterogeneity in dopamine in vitro models. We validate this disease model by showing robust expression of PD GWAS genes and overlap with postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, we find perturbations in glycolysis, cholesterol metabolism, synaptic signaling, and ubiquitin-proteasomal degradation. Overall, our study reveals cell type-specific perturbations in human dopamine neurons, which will further our understanding of PD and have implications for cell replacement therapies.


Assuntos
Neurônios Dopaminérgicos/patologia , Modelos Biológicos , Doença de Parkinson/genética , Doença de Parkinson/patologia , Análise de Célula Única , Estresse Fisiológico , Transcriptoma/genética , Diferenciação Celular/genética , Respiração Celular , Colesterol/metabolismo , Montagem e Desmontagem da Cromatina , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo/genética , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glicólise/genética , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Fosforilação Oxidativa , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise de Regressão , Transdução de Sinais , Estresse Fisiológico/genética , Sinapses/metabolismo , Ubiquitina/metabolismo , Regulação para Cima/genética
9.
Nat Commun ; 10(1): 1817, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000720

RESUMO

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.


Assuntos
Autofagia/efeitos dos fármacos , Reposicionamento de Medicamentos , Felodipino/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Embrião não Mamífero , Felodipino/uso terapêutico , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Cultura Primária de Células , Suínos , Porco Miniatura , Resultado do Tratamento , Peixe-Zebra , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
10.
Stem Cell Reports ; 12(4): 757-771, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30905739

RESUMO

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.


Assuntos
Reprogramação Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Estudo de Associação Genômica Ampla , Animais , Biomarcadores , Sistemas CRISPR-Cas , Linhagem Celular , Dosagem de Genes , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Nat Commun ; 10(1): 87, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622252

RESUMO

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Topotecan/farmacologia , Topotecan/uso terapêutico
12.
Nat Commun ; 9(1): 5378, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30568163

RESUMO

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Cromatina/metabolismo , Epigênese Genética , Células HL-60 , Hematopoese , Humanos , Células K562 , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA
13.
Nat Biotechnol ; 2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30480667

RESUMO

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

14.
Drug Resist Updat ; 40: 17-24, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30439622

RESUMO

Glioblastoma is the most common and malignant form of brain cancer, for which the standard treatment is maximal surgical resection, radiotherapy and chemotherapy. Despite these interventions, mean overall survival remains less than 15 months, during which extensive tumor infiltration throughout the brain occurs. The resulting metastasized cells in the brain are characterized by chemotherapy resistance and extensive intratumoral heterogeneity. An orthogonal approach attacking both intracellular resistance mechanisms as well as intercellular heterogeneity is necessary to halt tumor progression. For this reason, we established the WINDOW Consortium (Window for Improvement for Newly Diagnosed patients by Overcoming disease Worsening), in which we are establishing a strategy for rational selection and development of effective therapies against glioblastoma. Here, we overview the many challenges posed in treating glioblastoma, including selection of drug combinations that prevent therapy resistance, the need for drugs that have improved blood brain barrier penetration and strategies to counter heterogeneous cell populations within patients. Together, this forms the backbone of our strategy to attack glioblastoma.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Sistemas de Liberação de Medicamentos , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/efeitos adversos
15.
Nat Genet ; 50(6): 883-894, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29736013

RESUMO

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição GATA/genética , Histona Desmetilases/genética , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Histonas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Ativação Transcricional
16.
Prog Mol Biol Transl Sci ; 152: 49-67, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29150004

RESUMO

The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research, enabling genetic engineering of any gene in any cell, tissue, organ, and organism. The ability for fast, precise, and efficient profiling of the double-stranded break induced insertions and deletions (indels), mediated by any of the available programmable nucleases, is paramount to any given gene targeting approach. In this study we review the most commonly used indel detection methods and using a robust, sensitive, and cost efficient Indel Detection by Amplicon Analysis method, we have investigated the impact of the most commonly used CRISPR/Cas9 delivery formats, including lentivirus transduction, plasmid lipofection, and ribo nuclear protein electroporation, on the dynamics of indel profile formation. We observe rapid indel formation using RNP electroporation, especially with synthetic stabilized gRNA, as well as long-term decline in overall indel frequency with lipofectamine-based, plasmid transfection methods. Most methods reach peak editing on day 2-3 postdelivery. Furthermore, we find relative increase in frequency of larger size indels (>6bp) under condition of persistent editing using stably integrated lentiviral gRNA and Cas9 vectors.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mutação INDEL , Reparo do DNA , Humanos
17.
Nat Commun ; 8(1): 933, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038581

RESUMO

Mesodiencephalic dopamine neurons play central roles in the regulation of a wide range of brain functions, including voluntary movement and behavioral processes. These functions are served by distinct subtypes of mesodiencephalic dopamine neurons located in the substantia nigra pars compacta and the ventral tegmental area, which form the nigrostriatal, mesolimbic, and mesocortical pathways. Until now, mechanisms involved in dopaminergic circuit formation remained largely unknown. Here, we show that Lmx1a, Lmx1b, and Otx2 transcription factors control subtype-specific mesodiencephalic dopamine neurons and their appropriate axon innervation. Our results revealed that the expression of Plxnc1, an axon guidance receptor, is repressed by Lmx1a/b and enhanced by Otx2. We also found that Sema7a/Plxnc1 interactions are responsible for the segregation of nigrostriatal and mesolimbic dopaminergic pathways. These findings identify Lmx1a/b, Otx2, and Plxnc1 as determinants of dopaminergic circuit formation and should assist in engineering mesodiencephalic dopamine neurons capable of regenerating appropriate connections for cell therapy.Midbrain dopaminergic neurons (mDAs) in the VTA and SNpc project to different regions and form distinct circuits. Here the authors show that transcription factors Lmx1a, Lmx1b, and Otx2 control the axon guidance of mDAs and the segregation of mesolimbic and nigrostriatal dopaminergic pathways.


Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Fatores de Transcrição/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Axônios/fisiologia , Neurônios Dopaminérgicos/metabolismo , Feminino , Regulação da Expressão Gênica , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Fatores de Transcrição/genética , Área Tegmentar Ventral/fisiologia
18.
Sci Rep ; 7(1): 2244, 2017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-28533524

RESUMO

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Estudo de Associação Genômica Ampla , Animais , Proteínas Ligadas por GPI/metabolismo , Edição de Genes/métodos , Biblioteca Gênica , Vetores Genéticos , Estudo de Associação Genômica Ampla/métodos , Humanos , Lentivirus/genética , Camundongos , Fenótipo , RNA Guia , Transdução de Sinais
19.
Nat Genet ; 49(5): 730-741, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28319090

RESUMO

The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer. We validated ZBTB20, CELF2, PARD3, AKAP13 and WAC, which were identified by our screens in multiple cancer types, as new tumor suppressor genes in prostate cancer. We demonstrated their synergy with PTEN in preventing invasion in vitro and confirmed their clinical relevance. Further characterization of Wac in vivo showed obligate haploinsufficiency for this gene (which encodes an autophagy-regulating factor) in a Pten-deficient context. Our study identified complex PTEN-cooperating tumor suppressor networks in different cancer types, with potential clinical implications.


Assuntos
Elementos de DNA Transponíveis/genética , Genes Supressores de Tumor , Mutagênese Insercional , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Animais , Linhagem Celular , Movimento Celular/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Dosagem de Genes , Predisposição Genética para Doença/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos Knockout , Camundongos Transgênicos , Mutação , Próstata/citologia , Próstata/metabolismo , Interferência de RNA , Transdução de Sinais/genética
20.
Cell Rep ; 17(4): 1193-1205, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760321

RESUMO

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Terapia de Alvo Molecular , Adulto , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Humanos , Reprodutibilidade dos Testes
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