Core D: CRISPRi/a Core

项目总结 CRISPRi/a核心将通过支持击倒和过度表达来支持项目1、2和3的研究 人类IPSC来源神经元中内源性基因的研究。CRISPRi/a技术，我们共同- 开发，使高度特异的，可诱导的和可逆的哺乳动物基因表达控制 细胞。我们使用细菌Cas9蛋白的催化失活版本(DCas9)来招募转录 内源基因的抑制因子(CRISPRi)或转录激活因子(CRISPRa)，由 单引导RNA(SgRNAs)。我们已经通过两种模式确定了这项技术的使用：调查 感兴趣的单个基因的功能(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)。我们将支持研究 项目1、2和3，通过启用基于CRISPRi/a的人类IPSC来源的正向和反向遗传学 神经元。首先，我们将从等位基因的人类中建立并验证稳定的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IPSCs。然后，我们将生成和 验证靶向轴突初始片段(AIS)蛋白的sgRNAs，以便于研究其对 V337M tau神经元的可塑性和兴奋性(项目1)，针对关键自噬途径的sgRNAs 探讨调制这些通路是否能恢复V337M tau神经元的兴奋性 (对于项目1、2和3)，以及靶向与V337M tau选择性相互作用的蛋白质的sgRNAs 病原菌播种引起的神经元活动和自噬通路异常(For 项目1和项目3)。我们还将进行两次全基因组CRISPRi筛查。首先，我们将致力于确定 控制tau摄取的细胞途径，这将在项目2中进一步描述。第二，我们将 目的确定控制模板tau聚集的细胞通路。这些先进的遗传学方法将 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，目标是确定来自蛋白质组和遗传方法的趋同结果，并与 人类核心，以验证我们基于细胞的研究结果在人类患者中的相关性。

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.