Core D: CRISPRi/a Core

核心 D：CRISPRi/a 核心

• 批准号: 10011935
• 负责人: Martin Kampmann
• 金额: $ 18.03万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011935
• 关键词: Address; Autophagocytosis; Axon; Biological Assay; Biological Process; CRISPR interference; Cell Line; Cells; Communities; Complement; Data; Data Set; Diamond; Exposure to; Flow Cytometry; Fluorescence Resonance Energy Transfer; Gene Activation; Gene Expression; Genes; Genetic; Genetic Epistasis; Goals; Guide RNA; Human; Individual; Investigation; Label; Link; Mammalian Cell; Monitor; Neurons; Pathogenicity; Pathway interactions; Patients; Physiological; Proteins; Proteomics; Recombinants; Reporting; Research Project Grants; Research Support; Surveys; Tauopathies; Technology; Transcription Coactivator; Transcription Repressor; Work; base; combinatorial; forward genetics; genetic approach; genome wide screen; genome-wide; induced pluripotent stem cell; interest; knock-down; monomer; neuronal excitability; new therapeutic target; overexpression; proteostasis; recruit; reverse genetics; success; synergism; targeted biomarker; tau Proteins; tau aggregation; tool; uptake

PROJECT SUMMARY The CRISPRi/a core will support research of Projects 1, 2, and 3 by enabling knockdown and overexpression of endogenous genes in human iPSC-derived neurons. The CRISPRi/a technology, which we co- developed, enables highly specific, inducible and reversible control of gene expression in mammalian cells. We use a catalytically inactive version of the bacterial Cas9 protein (dCas9) to recruit transcriptional repressors (for CRISPRi) or transcriptional activators (for CRISPRa) to endogenous genes, as directed by single guide RNAs (sgRNAs). We have established the use of this technology in two modes: to investigate the function of individual genes of interest (reverse genetics), and to conduct genome-wide screens to uncover genes relevant for a biological process of interest (forward genetics). We will support the research of Projects 1, 2, and 3 by enabling CRISPRi/a-based forward and reverse genetics in human iPSC-derived neurons. First, we will generate and validate stable CRISPRi and CRISPRa cell lines from the isogenic human iPSCs expressing wild-type tau or V337M tau that are used by Projects 1, 2, and 3. Then, we will generate and validate sgRNAs targeting axon initial segment (AIS) proteins to enable the investigation of their effects on plasticity and excitability of V337M tau neurons (for Project 1), sgRNAs targeting key autophagy pathways to address the question if modulation of these pathways can restore neuronal excitability of V337M tau neurons (for Projects 1, 2, and 3), and sgRNAs targeting proteins selectively interacting with V337M tau that could underlie the abnormality in neuronal activity and autophagy pathways induced by pathogenic seeding (for Projects 1 and 3). We will also conduct two genome-wide CRISPRi screens. First, we will aim to identify cellular pathways controlling tau uptake, which will then be further characterized by Project 2. Second, we will aim to identify cellular pathways controlling templates tau aggregation. These forward genetics approaches will complement the hypothesis-driven reverse-genetics approaches and provide an unbiased survey of relevant cellular pathways. We will work with the Data core to integrate our datasets with those generated by the MS core, with the goal to identify convergent results from the proteomic and genetic approaches, and to work with the Human core to validate the relevance of our cell-based findings in human patients.

项目摘要 CRISPRi/a核心将通过实现敲除和过表达来支持项目1、2和3的研究。 内源性基因在人类iPSC衍生的神经元中的作用。CRISPRi/a技术，我们合作， 开发，使高度特异性，诱导和可逆的控制基因表达在哺乳动物 细胞我们使用细菌Cas9蛋白（dCas 9）的催化失活版本来募集转录 抑制子（对于CRISPRi）或转录激活子（对于CRISPRa）对内源基因的作用，如由 单向导RNA（sgRNA）。我们已经建立了两种模式的使用这种技术：调查 目的基因的功能（反向遗传学），并进行全基因组筛选， 与感兴趣的生物过程相关的基因（正向遗传学）。我们将支持以下研究： 项目1、2和3通过在人类iPSC衍生的细胞中实现基于CRISPRi/a的正向和反向遗传学， 神经元首先，我们将从同基因人类CRISPRi和CRISPRa细胞系中产生并验证稳定的CRISPRi和CRISPRa细胞系。 表达野生型tau或V337 M tau的iPSC，用于项目1、2和3。然后，我们将生成和 验证靶向轴突起始片段（AIS）蛋白的sgRNA，以研究它们对 V337 M tau神经元的可塑性和兴奋性（项目1），靶向关键自噬途径的sgRNA， 解决这些通路的调节是否可以恢复V337 M tau神经元的神经元兴奋性的问题 (for项目1、2和3），以及靶向与V337 M tau选择性相互作用的蛋白质的sgRNA，其可以 是病原性接种诱导的神经元活动和自噬途径异常的基础（对于 项目1和3）。我们还将进行两次全基因组CRISPRi筛选。首先，我们将确定 控制tau摄取的细胞途径，然后将由项目2进一步表征。二是 目的是鉴定控制模板tau聚集的细胞途径。这些遗传学方法将 补充假设驱动的反向遗传学方法，并提供一个公正的调查， 细胞通路我们将与数据核心合作，将我们的数据集与MS生成的数据集集成在一起。 核心，目标是从蛋白质组学和遗传学方法中确定趋同结果，并与 人类核心，以验证我们在人类患者中基于细胞的发现的相关性。