Systematic functional dissection of neuronal transcriptome diversity

神经元转录组多样性的系统功能剖析

基本信息

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
项目类别：
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

项目摘要

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）极大地增强了神经元转录组的多样性。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已被证实。我们率先开发了“以 RBP 为中心”的策略来控制神经元特异性剪接。使用综合方法精确重建特定类别神经 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。优先生成的发育调控外显子列表将被详细研究以改进我们的了解 AS 在神经分化各个阶段的功能重要性。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为了填补这一空白，我们建议使用外显子开发“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对于神经发育过程中的变异，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在目标1中，我们将建立一个大规模的基因组编辑平台来删除胚胎干细胞。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 个优先神经替代外显子进行试点筛选，并识别这些外显子根据神经形态学分析进行此筛选对于轴突生长很重要。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这些带有个体突变的克隆系将被以高通量形式转导。受并行神经分化、高通量成像和表型分析的影响。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，这将是因此，我们的方法将结合可扩展性和可扩展性的优势。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水平。这种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。