A CRISPR-Cas9 screen to identify genetic modifiers of APP/BACE-1 interactions

用于鉴定 APP/BACE-1 相互作用的遗传修饰剂的 CRISPR-Cas9 筛选

• 批准号: 9074668
• 负责人: Subhojit Roy
• 金额: $ 22.31万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9074668
• 关键词: Abeta synthesis; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Attenuated; Biological Assay; Brain; CRISPR screen; CRISPR/Cas technology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; DNA Double Strand Break; Endocytosis; Endosomes; Ensure; Enzymes; Event; Figs - dietary; Fluorescence; Frequencies; Gene Proteins; Gene Silencing; Genes; Genetic; Goals; Guide RNA; Hippocampus (Brain); Human; Human Genome; Image; Israel; Knock-out; Lead; Letters; Libraries; Life; Methods; Molecular; Nature; Neurons; Optics; Paper; Pathogenesis; Pathologic; Pathway interactions; Physiological; Play; Protein Fragment; Proteins; Proteolysis; Proxy; RNA Interference; Recycling; Reporting; Research; Role; Running; Science; Seminal; Site; Sorting - Cell Movement; Specificity; Students; System; Technology; Validation; Vesicle; base; beta-site APP cleaving enzyme 1; endonuclease; enzyme substrate; gene discovery; genome-wide; induced pluripotent stem cell; insight; loss of function; meetings; new therapeutic target; novel; protein expression; protein protein interaction; public health relevance; research study; screening; secretase; stem; tool; trafficking

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to discover molecules along trafficking pathways leading to the convergence of two key proteins in Alzheimer's disease (AD) pathogenesis - Amyloid Precursor Protein (APP) and β-site APP-cleaving enzyme-1 (BACE-1). This convergence, and consequent enzymatic β-cleavage of APP, is the rate-limiting step of amyloid beta (Aβ) production - a pathological hallmark of AD brains and a prevailing focus in AD research. Visualizing APP/BACE-1 trafficking in hippocampal neurons, we recently found that after synthesis, APP and BACE-1 are sorted into distinct vesicles, with BACE-1 selectively routed into recycling endosomes. At steady state, APP and BACE-1 convergence is a low-frequency event - producing Aβ at basal levels (Das et al., Neuron 2013; PMID: 23931995). Following up on these studies, we reasoned that ascertaining molecular pathways leading up-to this seminal convergence event would allow: 1) identification of the repertoire of trafficking pathways by which APP and BACE-1 meet to initiate the amyloidogenic cascade; and 2) discovery of novel "druggable targets" that can be manipulated to diminish APP/BACE-1 convergence and Aβ production. Towards this we developed an in-cellulo Optical assay to visualize Convergence of APP and BACE-1 (OptiCAB). Based on fluorescence complementation, this assay reports APP/BACE-1 interactions as a simple on/off readout, correlates with APP β-cleavage, and is suitable for large-scale analyses. Combining this assay with a newly-developed powerful genome-scale screen using CRISPR-Cas9 knockout (GeCKO) library (collaboration with Feng Zhang, MIT), our goal is to discover genes involved in `trafficking-related' upstream pathways that eventually lead to APP/BACE-1 convergence and Aβ production. Notably, CRISPR-Cas9- based screens are not limited by incomplete protein depletion and confounding off-target effects that have historically limited the utility of RNAi. Secondary validation of `hits' (i.e. genes that attenuate APP/BACE-1 interactions) will be done in human induced pluripotent stem cells (iPSC's); where APP-cleavage products will be analyzed after relevant CRISPR-knockout. Our aims are: Aim #1: Discover pathways leading to APP and BACE-1 convergence using OptiCAB and GeCKO; and Aim #2: Validate `hits' from Aim 1 in human neuronally-differentiated iPSCs. Our experiments will not only provide insights into the physiologic "amyloid-pathway" in humans, but may also offer new targets for AD. Finally, note that our focus on the repertoire of trafficking pathways leading up-t APP/BACE-1 approximation stems from our own live-imaging studies; and is different from the current narrow focus on enzymatic activity of the secretases.

 描述（由适用提供）：该提案的总体目标是沿运输途径发现分子，从而导致阿尔茨海默氏病（AD）发病机理中两种关键蛋白的收敛 - 淀粉样蛋白（APP）和β-site-Site App-app-app-app-cleace-app-cleace-app-app-cleace-app-app-app-app-app-app-app-app-app-app-app-app-app-app-cleaving酶 - 酶-1（bace-1）。 APP的这种收敛性和随之而来的酶促β-切解是淀粉样蛋白β（Aβ）生产的限制步骤，这是AD大脑的病理标志，并且是AD研究的主要重点。在海马神经元中可视化应用程序/BACE-1运输，我们最近发现合成后，App和Bace-1被分类为不同的蔬菜，而BACE-1选择性地将回收式内体路由。在稳定状态下，APP和BACE-1收敛是一个低频事件 - 在基本水平上产生Aβ（Das等，Neuron，2013； PMID：23931995）。在进行这些研究之后，我们认为确定导致第二次收敛事件的分子途径将允许：1）鉴定App和Bace-1符合淀粉样蛋白源性级联的运输途径的库存； 2）发现可以操纵的新型“可药物目标”，以减少APP/BACE-1收敛和Aβ产生。为此，我们开发了一种内部光学测定法，以可视化APP和BACE-1（OPTICAB）的收敛性。基于荧光完成，该测定法将APP/BACE-1相互作用报告为简单的ON/OFF读数，与APPβ-裂解相关，适用于大规模分析。使用CRISPR-CAS9淘汰赛（Gecko）库（与MIT的Feng Zhang合作）将此测定与新开发的强大基因组尺度屏幕相结合，我们的目标是发现与“贩运相关的”上游途径涉及的基因，有时会导致App/Bace-1 Convergence和Aβ产生。值得注意的是，基于CRISPR-CAS9的筛选不受蛋白质耗竭的不完整限制，并混淆了脱靶效应，这些效果一直限制了RNAi的效用。 “命中”的次要验证（即减弱App/bace-1相互作用的基因） 将在人类诱导的多能干细胞（IPSC）中完成；在相关的CRISPR-KNOCKOUT之后，将分析应用程序切换产品。我们的目标是：AIM＃1：使用Opticab和Gecko发现导致APP和BACE-1收敛的途径； AIM＃2：在人类神经差异的IPSC中验证AIM 1的“命中”。我们的实验不仅将提供有关人类生理“淀粉样蛋白探针”的见解，而且还可以为AD提供新的目标。最后，请注意，我们关注的是，从我们自己的现场模仿研究中，导致UP-T App/Bace-1近似步骤的贩运途径的曲目；并且与当前对分泌酶的酶活性的狭窄关注不同。