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.

项目概要 神经元转录组多样性的系统功能剖析 细胞类型特异性选择性剪接（AS）极大地放大了神经元转录组的多样性。恰当的 这种分子复杂性的调节及其在发育过程中的建立对于成熟至关重要 神经细胞及其稳态的维持。多种 RNA 结合蛋白 (RBP) 确定控制神经元特异性剪接。我们率先制定了“以 RBP 为中心”的战略 使用综合方法精确重建特定类别神经元 RBP 的剪接调节网络 结合了多种实验和计算数据模式的分析框架。这些努力 生成了发育调控外显子的优先列表，将对其进行详细研究以改进我们的 了解 AS 在神经元分化的各个阶段的功能重要性。然而，一个 该领域的主要障碍是我们目前无法有效地询问大量的功能 剪接变体。为了填补这一空白，我们建议使用外显子来开发“以外显子为中心”的策略 特异性遗传筛选，直接系统地剖析特定剪接的功能作用 神经发育过程中的变异。对于试点研究，我们的重点是确定替代外显子 在小鼠脊髓运动神经元体外模型系统中调节轴突形态发生 胚胎干（mES）细胞。目标1，我们将建立一个大规模的基因组编辑平台，删除 通过基于慢病毒、CRISPR/Cas9 介导的基因组在 mES 细胞中单独替代外显子 工程。我们设计了一种克隆策略，使我们能够构建具有大量库的 CRISPR 文库。 配对引导 RNA (gRNA) 靶向单个替代外显子以触发特定的外显子删除。参数 为了优化文库的复杂性，将建立病毒传递和基因组编辑的效率。 在目标 2 中，我们将对约 100 个优先神经元替代外显子进行试点筛选，并识别这些外显子 对于轴突生长很重要。根据神经元形态分析进行此筛选 由于缺乏可靠的报告基因，我们提出了一种从 mES 细胞群中衍生克隆突变体的策略 以高通量形式转导的细胞池。这些携带个体突变的克隆系将被 进行并行神经元分化、高通量成像和表型分析。这个策略 将允许灵敏地检测在轴突生长中表现出精细形态缺陷的突变体，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性和 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认识扩展到 各个剪接变体。该策略可以很容易地用于研究潜在的分子程序 正常和病理情况下的神经分化、迁移和功能。