Supplement for Center for Synthetic Regulatory Genomics: Building CACNA1C alleles associated with Neuropsychiatric Disorders

项目摘要 合成调控基因组学中心（SyRGe）的任务是开发和应用 对大基因位点进行协调改变的技术，广泛地使研究功能成为可能。 全基因组关联研究（GWAS）“命中”揭示的调控序列。中心的工具 显著地取代了目前用于操纵和评估调控基因组功能的技术 通过专注于所谓的大DNA的组装和交付。本补充请求提议， 利用我们在大DNA组装和交付方面的最新技术发展， CACNA 1C，包含与各种神经精神疾病相关的最高命中率，包括精神分裂症， 双相情感障碍，重度抑郁症，多动症和自闭症。CACNA 1C编码L型的孔形成亚基 电压门控钙通道，广泛表达于神经元、内分泌细胞、心肌细胞和 平滑肌通过通道的钙内流对于突触可塑性、基因表达是至关重要的 调节、胰岛素分泌和肌肉收缩。在这一补充请求中，我们建议组装和 递送~330 kb人CACNA 1C内含子3以替换小鼠内源性Cacna 1c内含子3。我们将 构建并递送含有风险或保护性等位基因的人CACNA 1C内含子3， 将小鼠胚胎干细胞改造成神经元用于初始表型分析。我们还将构建一系列变体， 功能研究。我们将评估分化神经元中的Cacna 1c mRNA和L型钙电流。 本补充请求中的拟议工作将使人们能够深入了解 多种神经精神疾病，它也将作为进一步产生人性化的基础。 用于行为研究的Cacna 1c小鼠模型。

Project Summary The Center for Synthetic Regulatory Genomics (SyRGe) is tasked with development and application of technology for making coordinated changes to large gene loci, broadly enabling investigations into the function of regulatory sequences revealed by genome wide association study (GWAS) “hits”. The Center’s tools dramatically supersede present technologies for manipulation and assessment of regulatory genome function through its focus on assembly and delivery of so-called Big DNA. This supplement request proposes to leverage our most recent technology development in Big DNA assembly and delivery to tackle a large locus in CACNA1C, containing the top hits associated with various neuropsychiatric disorders, including schizophrenia, bipolar disorder, major depression, ADHD and ASD. CACNA1C encodes a pore-forming subunit of the L-type voltage-gated calcium channel and is widely expressed in neurons, endocrine cells, cardiomyocytes and smooth muscles. Calcium influx through the channel is critical for synaptic plasticity, gene expression regulation, insulin secretion and muscle contraction. In this supplement request, we propose to assemble and deliver the ~330 kb human CACNA1C intron 3 to replace the mouse endogenous Cacna1c intron 3. We will build and deliver human CACNA1C intron 3 that contains risk or protective alleles and differentiate the engineered mouse ESCs into neurons for initial phenotyping. We will also build a series of variants for further functional studies. We will assess the Cacna1c mRNA and L-type calcium current in differentiated neurons. The proposed work in this supplement request will enable insights into the molecular mechanisms underlying multiple neuropsychiatric disorders, and it will also serve as the foundation for further generation of humanized Cacna1c mouse models for behavioral studies.