Massively-parallel functional interrogation of psychiatric genetics

 描述(申请人提供)：人类基因组测序的快速发展产生了大量的数据，因此产生了大量的假设。对于精神疾病，一些里程碑式的研究已经产生了与特定疾病有关的基因变异清单。然而，由于缺乏合适的模型系统和高通量的功能分析，对这些假设的功能测试仍然具有挑战性。在这里，我们建议开发和应用一系列高通量基因组扰动方法，以实现从大规模遗传学数据中快速识别因果遗传变异。我们提议开发的技术将具有非常广泛的适用性，并有可能从根本上改变不同生物医学领域的发现规模和速度。作为这项提议的一部分，将开发三项补充技术：1.功能筛选方法，以便能够快速确定特定细胞类型的重新编程因素。通过使用基因组规模的转录激活或表观遗传重编程文库，我们将建立一种系统的方法来反向工程与不同神经精神疾病相关的神经元亚型的重编程因子的充分组合，例如精神分裂症中的小白蛋白阳性中间神经元。2.大规模和系统的基因组扰动工具，以实现对遗传变异的大规模平行功能测试，从而能够将相关遗传变异的长清单缩小到短的因果变异清单。我们将建立基因扰动技术，对编码和非编码基因组区域进行多重和系统的缺失或突变分析，并探测它们的表观遗传状态，以确定因果遗传变异。3.高效和精确的基因组修改技术，能够将因果变异迅速引入细胞或动物模型，从而能够阐明其致病机制。虽然基于核酸酶的基因组编辑系统能够有效地敲除基因，但基因插入或基因校正仍然效率低下。我们将描述一种新的程序化基因组重排机制，并将其发展为一种高效和精确的哺乳动物基因组编辑技术。鉴于这项技术的广泛适用性，这将显著加快细胞和动物模型的生成，这项拟议工作的影响将是深远的，并将从根本上改变现有的研究生命科学和医学所有领域基因相互作用的实验方法。

 DESCRIPTION (provided by applicant): The rapid advances in human genome sequencing has generated a large amount of data and consequently a large number of hypotheses. For psychiatric disease, a number of landmark studies have led to lists of genetic variations that are linked to specific diseases. However, functional testing of these hypotheses has remained challenging due to a lack of appropriate model systems and high-throughput functional assays. Here we propose to develop and apply a series of high-throughput genome perturbation methodologies to enable the rapid identification of causal genetic variants from large-scale genetics data. The technology we propose to develop will be very broadly applicable and has the potential to radically transform the scale and rate of discovery across different biomedical fields. Three complementary technologies will be developed as a part of this proposal: 1. Functional screening methods to enable rapid identification of reprogramming factors for specific cell types. By using genome-scale transcription activation or epigenetic reprogramming libraries, we will establish a systematic approach to reverse-engineer the sufficient combinations of reprogramming factors for neuron subtypes that are relevant for different neuropsychiatric diseases, such as parvalbumin positive interneurons in schizophrenia. 2. Large-scale and systematic genome perturbation tools to enable massively-parallel functional testing of genetic variants, to enable narrowing of the long lists of correlated genetic variants to a short list of causal variants. We will establish genetic perturbation technologies to enable multiplex and systematic deletion or mutational analysis of coding and non-coding genomic regions, as well as probing of their epigenetic states, to identify causal genetic variants. 3. Efficient and precise genome modification technologies to enable rapid introduction of causal variants into cellular or animal models to enable elucidation of their disease-causing mechanisms. Whereas nuclease-based genome editing systems enables efficient gene knockout, gene insertion or gene correction remains inefficient. We will characterize a novel programmed genome rearrangement mechanism and develop it into a technology for efficient and precise mammalian genome editing. This will significantly accelerate the generation of cellular and animal models Given the broad applicability of this technology, the impact of this proposed work will be far reaching and will radically transform existing experimental approaches for studying gene interactions in all fields of life science and medicine.