Dissecting recurrent microdeletion syndromes using dual-guide genome editing

使用双引导基因组编辑剖析复发性微缺失综合征

基本信息

批准号：
9087365
负责人：
JAMES F GUSELLA
金额：
$ 58.08万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9087365
关键词：
16p11.2 1q21 22q11.2 Ablation Biological Assay Biological Markers CRISPR/Cas technology Cell Line Cells Critical Pathways Data Development Disease Etiology Failure Future Gene Expression Gene Expression Profile Gene Mutation Gene Silencing Genes Genetic Genetic Predisposition to Disease Genome Genomics Global Change Haploidy Health Hereditary Disease Human Genome Individual Mediating Mental disorders Molecular Mutation Nervous system structure Neurodevelopmental Disorder Neuronal Differentiation Neurons Outcome Overlapping Genes Pathway interactions Patients Phenotype Process Recurrence Route Series Syndrome System Technology Testing Therapeutic Intervention autism spectrum disorder developmental disease differential expression dosage genome editing homologous recombination induced pluripotent stem cell innovation knockout gene microdeletion mutant nerve stem cell neurodevelopment novel overexpression targeted treatment transcriptome transcriptome sequencing

项目摘要

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.

描述（由申请人提供）：复发性微缺失综合征 (rMDS)，例如与 16p11.2、1q21 和 22q11.2 相关的综合征，是神经发育障碍（包括自闭症谱系）遗传病因的主要组成部分 (10-15%)在每个 rMDS 中，由于非等位基因，一组不同的基因可重复地减少为单倍体剂量。由侧翼节段重复介导的同源重组 (NHAR) 理论上，rMDS 的神经发育表型可能主要源自单个基因驱动因素，正如我们所证明的非复发性 MDS 的情况，或者源自多个基因的联合效应。该项目将测试这三种常见 rMDS 的转录效应集中在对异常神经发育至关重要的少数途径/过程的假设。负责与这些 rMDS 相关的失调网络的个体遗传驱动因素，以及是否可以通过直接将驱动基因重新引入细胞系统或通过反式拯救来挽救发生的转录变化该项目将利用我们在 CRISPR/Cas9 基因组编辑方面的最新进展，使用新型双引导方法有效地在诱导多能干细胞 (iPSC) 中产生大量缺失，这些同基因细胞消除了不同遗传背景的混乱。具体来说，我们的目标是比较这三个 rMDS 区域中完全缺失的转录后果，并识别由于每个完全微缺失的单倍体不足而改变的重叠基因/通路。（目标 1）然后，我们将通过 rMDS 区域内的系统单基因消融来寻求识别每个 rMDS 中的关键遗传驱动因素（目标 2），我们将测试是否可以通过重新引入单个驱动因素来挽救关键的网络改变。基因或操纵失调网络中的基因中心（目标 3）最终，本研究将解构三种最常见的 rMDS，以产生可以通过二次修复的关键神经发育相关网络。操纵，从而为发现神经生物标志物和有针对性的治疗干预提供直接途径。