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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.

项目总结