Enhancer-targeted correction of haploinsufficient autism risk genes

项目摘要/摘要 自闭症谱系障碍(ASD)是一种高度异质性和高度遗传性的疾病，具有复杂的遗传 然而，大约20%的遗传风险是由主要基因突变的从头开始或新出现的 效果。这些基因中的大多数突变要么是已知的，要么是预测会导致截短的mRNA和 蛋白质产物，强烈表明可能的单倍性不足，其中功能基因的单个拷贝不是 足以使大脑正常发育或发挥作用的。我提出了一种谨慎的阶段性方法来纠正这些影响 通过激活非突变拷贝的表达来恢复正常的基因表达 级别。我将利用基因编辑技术的进步-使用CRISPR-A靶向高表达的增强子区域 在基于干细胞的3D皮质球体(HCS)的体外信心ASD基因，这已被证明 概括了活体皮质发育的许多特征。CRISPR-A具有以下显著优势 利用内源性基因表达调控元件，而无需编辑基因组或依赖人工 过度表达的基因构建。我还利用之前的工作，在这些工作中，我们创造了高度的信心 基因组范围的增强子图谱将这些转录激活区与它们的同源基因连接起来。我 建议：i)对12个高置信度ASD基因的假定增强子序列进行功能验证，ii)测试 选择HCS中的增强剂来表征它们对神经元分化的影响，以及III)表征变化 在携带ASD风险突变的HCS的发育和形态表型上，与野生型HC相比， 并测定CRISPR-A在突变体中挽救基因表达和恢复野生型发育的能力 HCS。我将利用各种技术，包括单细胞测序和清晰度成像，来 全面显示皮质球体内神经元亚型的发育。这项研究的结果 可能不仅提供了基因激活作为一种治疗干预的原则证据，而且实质上 加深对ASD基因单倍性不足的影响及其神经生物学的了解 它们影响神经元发育的机制。

Project Summary/Abstract Autism Spectrum Disorder (ASD) is a highly heterogeneous and highly heritable disease with complex genetic contributions, and yet about 20% of genetic risk is imparted by de novo, or newly arising, mutations of major effect. The majority of mutations in these genes are either known to or predicted to lead to truncated mRNA and protein products, strongly indicating likely haploinsufficiency, in which a single copy of a functional gene is not sufficient for normal brain development or function. I propose a carefully staged approach to correct the effects of these mutations by activating expression from the non-mutant copy to restore gene expression to normal levels. I will leverage advances in gene editing technology - using CRISPR-A to target enhancer regions of high confidence ASD genes in stem cell-based 3D cortical spheroids (hCS) in vitro, which have been shown to recapitulate many features of in vivo cortex development. CRISPR-A has the significant advantages of leveraging endogenous gene expression regulatory elements without editing the genome or relying on artificial gene constructs for overexpression. I also leverage previous work where we have created high-confidence genome-wide enhancer maps that connect these transcriptional activating regions with their cognate genes. I propose to: i) functionally validate putative enhancer sequences for 12 high confidence ASD genes, ii) test selected enhancers in hCS to characterize their effect on neuronal differentiation, and iii) characterize changes in developmental and morphological phenotypes in hCS carrying ASD risk mutations compared to wildtype hCS, and determine the ability of CRISPR-A to rescue gene expression and restore wildtype development in mutant hCS. I will utilize diverse technologies, including single cell sequencing and CLARITY imaging, to comprehensively visualize the development of neuronal subtypes in cortical spheroids. Results from this study may not only provide a proof of principle for gene activation as a therapeutic intervention but will substantially enhance our understanding of the effects of haploinsufficiency in ASD genes and the neurobiological mechanisms whereby they impact neuronal development.