Developing a Pathway from Genetic Locus to Gene for Complex Traits in Rodents

在过去的20年里，已经鉴定出数千个遗传基因座，这些基因座导致了人类的复杂性状。 啮齿动物，包括常见疾病的模型。这些发现有望促进我们对 疾病的生物学机制和其他生物医学特征，但相对于 成功的映射实验产生的新见解是非常小的。这是因为映射 实验很少导致基因的识别。这项建议将从根本上改变这种情况， 采用创新的方法来识别基因位点上的基因，这些基因有助于复杂的 在老鼠身上。通过使用PI开发的资源和技术，包括使用远交种 高分辨率遗传作图的啮齿动物，基因组小鼠品系中遗传变异的目录 测序和基因鉴定方法，将开发一种有效和简单的方案， 使研究人员能够快速地从基因定位到基因识别。由于基因鉴定特别重要， 重要的（和具有挑战性的）精神疾病的模型，其中病因的理解仍然有限， 获得相关组织或细胞类型困难，该方法的功效在动物模型上进行了测试， 焦虑 我们的方法包括三个步骤：第一，确保支持每个位点的关联证据是稳健的; 第二，在每个位点上鉴定所有候选基因;第三，在近交系上敲除这些基因 并使用数量性状位点基因敲除相互作用测试来测试它们的候选资格。使用发现集， 62个基因座，有助于小鼠焦虑的变化，我们的目标是确定24个基因座与两个或更少的候选 基因，并确认在这些位点的焦虑相关基因的身份。直到最近， 使基因鉴定成为可能的相互作用测试，由于 在相同的遗传背景下获得敲除和野生型的困难。迈向新 基因工程技术CRISPR/Cas9克服了这一障碍。我们将利用这一点 使复杂性状位点的基因鉴定成为一项常规任务。我们的发现将改变复杂的 通过识别多达24个与焦虑有关的基因，将在啮齿类动物的遗传学方面取得重大进展。 了解一种常见疾病的生物学基础，从而对开发新的 治疗

Over the last 20 years many thousands of genetic loci have been identified that contribute to complex traits in rodents, including models of common diseases. The findings are expected to advance our understanding of biological mechanisms underlying disease and other traits of biomedical interest, yet relative to the number of successful mapping experiments the yield of novel insights is very small. This is because the mapping experiments have rarely led to the identification of genes. This proposal will radically change this situation by deploying an innovative approach to identifying genes at genetic loci that contribute to variation in complex traits in mice. By using resources and techniques that the PI has developed, including the use of outbred rodents for high-resolution genetic mapping, catalogs of genetic variants in mouse strains from genome sequencing, and methods for gene identification, an efficient and simple protocol will be developed that will allow researchers to rapidly progress from locus to gene identification. Since gene identification is particularly important (and challenging) in models of psychiatric disease where etiologic understanding is still limited and access to the relevant tissues or cell type difficult, the efficacy of the approach is tested on animal models of anxiety. Our approach consists of three steps: first, ensure that association evidence supporting each locus is robust; second, identify at each locus all candidate genes; third, make knockouts of those genes on an inbred strain and test their candidacy using a quantitative trait locus gene-knockout interaction test. Using a discovery set of 62 loci that contribute to variation in anxiety in mice, we aim to identify 24 loci with two or fewer candidate genes, and to confirm the identity of genes involved in anxiety at these loci. Until recently the key experiment that makes gene identification possible, the interaction test, could not easily be implemented because of the difficulty of obtaining a knockout and wildtype on the same genetic background. The advent of the new genomic engineering technology, CRISPR/Cas9, has overcome that obstacle. We will take advantage of this advance to make gene identification at complex trait loci a routine task. Our findings will transform complex trait genetics in rodents, and, by identifying up to 24 genes involved in anxiety, will make a major inroad into understanding the biological basis of a common disease, with consequent implications for developing new therapies.