Core D: CRISPRi/a Core

核心 D：CRISPRi/a 核心

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 CRISPRi/a 核心将通过实现敲低和过度表达来支持项目 1、2 和 3 的研究人类 iPSC 衍生神经元中的内源基因。我们共同开发的 CRISPRi/a 技术开发的，能够高度特异性、诱导性和可逆性地控制哺乳动物的基因表达细胞。我们使用细菌 Cas9 蛋白 (dCas9) 的催化失活版本来招募转录内源基因的阻遏子（对于 CRISPRi）或转录激活子（对于 CRISPRa），按照单引导RNA (sgRNA)。我们已经以两种模式建立了该技术的使用：单个感兴趣基因的功能（反向遗传学），并进行全基因组筛选以发现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中生成并验证稳定的 CRISPRi 和 CRISPRa 细胞系项目 1、2 和 3 使用的表达野生型 tau 或 V337M tau 的 iPSC。然后，我们将生成并验证靶向轴突初始片段 (AIS) 蛋白的 sgRNA，以便研究它们对 V337M tau 神经元的可塑性和兴奋性（项目 1），sgRNA 靶向关键自噬途径解决这些通路的调节是否可以恢复 V337M tau 神经元的神经元兴奋性的问题（针对项目 1、2 和 3），以及靶向与 V337M tau 选择性相互作用的蛋白质的 sgRNA 是致病性播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，我们的目标是确定控制 tau 摄取的细胞途径，然后将通过项目 2 进一步表征。其次，我们将旨在确定控制模板 tau 聚集的细胞途径。这些正向遗传学方法将补充假设驱动的反向遗传学方法，并提供相关的公正调查细胞途径。我们将与数据核心合作，将我们的数据集与 MS 生成的数据集集成核心，目标是确定蛋白质组学和遗传学方法的收敛结果，并与 Human core 来验证我们基于细胞的研究结果在人类患者中的相关性。