Systematic functional dissection of neuronal transcriptome diversity

神经元转录组多样性的系统功能剖析

• 批准号: 9272022
• 负责人: Chaolin Zhang
• 金额: $ 19.9万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272022
• 关键词: Address; Alpha Cell; Alternative Splicing; Autistic Disorder; Axon; Biological Assay; Biological Models; Brain; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cell Maintenance; Cell physiology; Cells; Clonal Expansion; Clone Cells; Cloning; Computational Technique; Data; Defect; Detection; Development; Developmental Process; Disease; Dissection; Employee Strikes; Epilepsy; Exons; Flow Cytometry; Genes; Genetic Screening; Genome engineering; Genotype; Guide RNA; Homeostasis; Image; Image Analysis; In Vitro; Individual; Knowledge; Libraries; Literature; Mammals; Mediating; Messenger RNA; Methods; Modality; Molecular; Monitor; Morphogenesis; Morphology; Motor Neurons; Mus; Mutant Strains Mice; Mutation; Nature; Neuraxis; Neuronal Differentiation; Neurons; Nonhomologous DNA End Joining; Pathologic; Phenotype; Physiology; Pilot Projects; Population; Process; Protein Isoforms; Proteome; Protocols documentation; RNA Splicing; RNA library; RNA-Binding Proteins; Regulation; Reporter; Role; Sorting - Cell Movement; Spinal; Subfamily lentivirinae; System; Systems Analysis; Testing; Transcript; Variant; Viral; Virus; Work; axon growth; base; candidate selection; cell type; cellular transduction; design; driving force; embryonic stem cell; experimental study; flexibility; gene function; genome editing; genome-wide; improved; in vitro Model; mammalian genome; migration; mutant; nervous system disorder; neurodevelopment; new therapeutic target; novel; particle; programs; relating to nervous system; screening; success; transcriptome

Project summary Systematic functional dissection of neuronal transcriptome diversity Cell type-specific alternative splicing (AS) enormously amplifies the neuronal transcriptome diversity. Proper regulation of such molecular complexity and its establishment during development is critical for the maturation of nerve cells and maintenance of their homeostasis. Multiple RNA-binding proteins (RBPs) have been identified to control neuron-specific splicing. We pioneered the development of an “RBP-centric” strategy to reconstruct precisely the splicing regulatory networks of specific classes of neuronal RBPs using an integrative analysis framework that combines multiple modalities of experimental and computational data. These efforts generated prioritized lists of developmentally regulated exons that will be studied in details to improve our understanding of the functional importance of AS at various stages of neuronal differentiation. However, a major roadblock for the field is our current inability to efficiently interrogate the function of a vast number of splice variants. To fill in this gap, we propose to develop an “exon-centric” strategy using an exon- specific genetic screen to dissect directly and systematically the functional role of specific splice variants during neural development. For a pilot study, our focus is to identify alternative exons that regulate axon morphogenesis in an in vitro model system of spinal motor neurons derived from mouse embryonic stem (mES) cells. In Aim 1, we will establish a large-scale genome-editing platform to delete individual alternative exons in mES cells through lentivirus-based, CRISPR/Cas9-mediated genome engineering. We designed a cloning strategy that will allow us to build a CRISPR library with a large pool of paired guide RNAs (gRNAs) targeting individual alternative exons to trigger specific exon deletion. Parameters for optimizing the complexity of the library, viral delivery, and efficiency of genome editing will be established. In Aim 2, we will perform a pilot screen of ~100 prioritized neuronal alternative exons and identify those important for axon outgrowth. To perform this screening based on analysis of neuronal morphology in the absence of a reliable reporter, we propose a strategy to derive clonal mutant mES cell populations from transduced cell pools in a high-throughput format. These clonal lines carrying individual mutations will be subject to paralleled neuronal differentiation, high-throughput imaging and phenotypic analysis. This strategy will allow sensitive detection of mutants showing fine morphological defects in axon growth, which will be genotyped and further validated. Our approach will therefore combine advantages of being both scalable and flexible. This study will establish a very effective method to extend our knowledge of gene function to the level of individual splice variants. This strategy can be readily adapted to study the molecular programs underlying neural differentiation, migration, and function in normal and pathological contexts.

项目摘要 神经元转录组多样性的系统功能解剖 细胞类型特异性的替代剪接（AS）极大的放大器神经​​元转录组多样性。恰当的 调节这种分子复杂性及其在开发过程中的建立对于成熟至关重要 神经细胞和维持其稳态。多种RNA结合蛋白（RBP）已经 确定可以控制神经元特异性剪接。我们开创了“以RBP为中心”策略的制定 精确地重建特定类别的神经元RBP的剪接调节网络 分析框架结合了实验和计算数据的多种方式。这些努力 生成的优先级列表的开发外显子将进行详细研究，以改善我们的 了解在神经元分化的各个阶段AS的功能重要性。但是， 该领域的主要障碍是我们目前无法有效审问大量的功能 剪接变体。为了填补这一空白，我们建议使用外显子制定“以外显子”策略 特定的遗传筛选，直接和系统地剖析特定剪接的功能作用 神经发育过程中的变体。对于试点研究，我们的重点是确定替代外显子 调节源自小鼠的脊柱运动神经元的体外模型系统中的轴突形态发生 胚胎（MES）细胞。在AIM 1中，我们将建立一个大规模的基因组编辑平台来删除 MES细胞中的单个替代外显子通过基于慢病毒的CRISPR/CAS9介导的基因组 工程。我们设计了一种克隆策略，该策略将使我们能够建立一个带有大量池 靶向各个替代外显子以触发特定外显子缺失的配对引导RNA（GRNA）。参数 为了优化文库的复杂性，将建立病毒式传递和基因组编辑效率。 在AIM 2中，我们将执行约100个优先神经元外显子的试验屏幕，并确定这些外显子 对于轴突生长至关重要。根据对神经元形态的分析进行此筛查 我们没有可靠的记者，我们提出了一种策略，以从 以高通量格式转导细胞池。这些带有个体突变的克隆线将是 进行平行的神经元分化，高通量成像和表型分析。这个策略 将允许对突变体的敏感检测在轴突生长中显示出细小的形态缺陷，这将是 基因分型并进一步验证。因此，我们的方法将结合可扩展和 灵活的。这项研究将建立一种非常有效的方法，将我们对基因功能的了解扩展到水平 单个剪接变体。可以很容易地适应此策略来研究依据的分子程序 在正常和病理环境中的神经分化，迁移和功能。