Dissecting recurrent microdeletion syndromes using dual-guide genome editing

 描述（由申请人提供）：复发性微缺失综合征（rMDS），如与16 p11.2、1 q21和22 q11.2相关的综合征，是神经发育障碍（包括自闭症谱系障碍（ASD））遗传病因学的主要组成部分（10-15%）。在每个rMDS中，由于侧翼节段重复介导的非等位基因同源重组（NHAR），一组不同的基因可重复地减少到单倍体剂量。rMDS中的神经发育表型理论上可能主要来源于单个基因驱动，正如我们已经证明的那样，通常是非复发性MDS的情况，或者来自多个基因的联合作用，这些基因共同失调。该项目将测试这三种常见的rMDS的转录效应收敛于对异常神经发育至关重要的少数通路/过程的假设。它将进一步评估是否存在负责与这些rMDS相关的失调网络的个体遗传驱动因子，以及是否可以通过直接将驱动因子基因重新引入细胞系统或通过转录因子的反式拯救来拯救发生的转录变化。 二级网络。该项目将利用我们在CRISPR/Cas9基因组编辑方面的最新进展，使用新型双指导方法在诱导多能干细胞（iPSC）中有效地产生大缺失。这些同基因细胞消除了不同遗传背景的混淆，从而提供了一个强大的测定系统。具体来说，我们的目标是比较在这三个rMDS区域的完全删除的转录后果，并确定重叠的基因/途径，由于每个完整的微缺失单倍不足改变（目的1）。然后，我们将通过rMDS区域内的系统性单基因消融来寻求确定每个rMDS内的关键遗传驱动因素（目的2）。最后，我们将测试是否可以通过重新引入单个驱动基因或操纵失调网络中的枢纽基因来挽救关键的网络改变（目标3）。在其结论中，这项研究将解构三种最常见的rMDS，以产生关键的神经发育相关网络，这些网络可以通过二次操作来挽救，从而为发现神经元生物标志物和靶向治疗干预提供了直接途径。

 DESCRIPTION (provided by applicant): Recurrent microdeletion syndromes (rMDS), such as those associated with 16p11.2, 1q21 and 22q11.2, represent a major component (10-15%) of the genetic etiology of neurodevelopmental disorders, including autism spectrum disorder (ASD). In each rMDS, a distinct set of genes is reproducibly reduced to haploid dosage due to non-allelic homologous recombination (NHAR) mediated by flanking segmental duplications. The neurodevelopmental phenotypes in rMDS could theoretically derive primarily from a single gene driver, as we have shown is often the case for non-recurrent MDS, or from the combined effects of multiple genes that are dysregulated in concert. This project will test the hypothesis that the transcriptional effects of these three common rMDS converge on a small number of pathways/processes that are critical for abnormal neurodevelopment. It will further evaluate whether there are individual genetic drivers responsible for the dysregulated networks associated with these rMDS, and whether the transcriptional changes that occur can be rescued either by directly re-introducing the driver gene into a cellular system or through trans-rescue of secondary networks. The project will capitalize on our recent advances in CRISPR/Cas9 genome editing to efficiently generate large deletions in induced pluripotent stem cells (iPSC) using a novel dual-guide approach. These isogenic cells eliminate the confound of differing genetic backgrounds and thereby provide a powerful assay system. Specifically, we aim to compare the transcriptional consequences of full deletion in these three rMDS regions and identify overlapping genes/pathways that are altered due to haploinsufficiency for each full microdeletion (Aim 1). We will then seek to identify key genetic drivers within each rMDS by systematic single gene ablation within the rMDS region (Aim 2). Finally, we will test whether critical network alterations can be rescued by re-introducing a single driver gene or manipulating hub genes within dysregulated networks (Aim 3). At its conclusion, this study will have deconstructed three of the most common rMDS to yield critical neurodevelopment-associated networks that can be rescued by secondary manipulation, thereby offering a direct route to the discovery of neuronal biomarkers and targeted therapeutic intervention.