Scalable in vivo tools for annotating and manipulating the druggable genome

描述：神经系统疾病是所有人类疾病中最具破坏性和最沉重的疾病之一，但它们仍然是治疗最差的疾病之一。对于大多数中枢神经系统疾病，我们对潜在的生物学知之甚少，并且尚未确定有效的治疗靶点。具有讽刺意味的是，基因组中最具药物药性的蛋白质类别在神经系统中具有高度代表性，并有助于神经系统疾病的病理。如果能更好地了解它们的神经系统功能，其中一些无疑将成为极好的治疗靶点。不幸的是，给这些特征不明确的基因分配功能仍然是相当具有挑战性的，特别是那些涉及中枢神经系统复杂工作的基因。以细胞为基础的体外试验完全不足以模拟神经系统中基因的功能。系统地破坏体内基因功能和广泛地评估对神经系统的影响需要可扩展的、负担得起的方法。斑马鱼提供了一个独特的机会来阐明可药物基因组的基因。在过去的五年里，新的工具使得对斑马鱼基因组进行有针对性的改变成为可能。同时，已经开发了12种新的行为分析方法，可以广泛地评估基因或小分子对神经系统的影响。本项目将这些基因组操作的新技术与高通量行为分析相结合，以表征可药物基因组的基因。研究人员将利用CRISPR-Cas系统敲除斑马鱼体内的相关基因，并对突变动物进行全面的行为分析，以评估其对神经系统的影响。在平行实验中，相同的基因将在斑马鱼基因组中被一种新的可视化/消融盒取代，从而实现体内表达模式的详细4D可视化。最后，一个独特的计算资源将使候选配体（探针）为感兴趣的蛋白质容易识别。这些概念验证实验将集中在15个基因上，其中包括特征良好和特征不佳的例子，并将为更全面地探索可药物基因组铺平道路。目标包括：目标1。表征可用药基因组中基因的神经系统功能。目标2。开发可视化和操纵可药物基因组基因的工具。目标3。鉴定可用药基因组中基因的候选配体。通过提供详细的敲除表型、体内表达模式和每个感兴趣基因的小分子探针，我们希望推进阐明可药物基因组基因的重要目标。

DESCRIPTION: Diseases of the nervous system are among the most devastating and burdensome of all human illnesses, yet they remain among the most poorly treated. For most CNS disorders, we understand the underlying biology poorly, and validated therapeutic targets have not been identified. Ironically, the most druggable protein classes in the genome are highly represented in the nervous system and contribute to the pathology of nervous system disorders. If their nervous system functions were better understood, some would undoubtedly be excellent therapeutic targets. Unfortunately, it remains quite challenging to assign functions to these poorly characterized genes, especially those involved in the complex workings of the CNS. Cell-based, in vitro assays are wholly inadequate to model the functions of genes in the nervous system. Scalable, affordable approaches are needed for systematically disrupting gene function in vivo and assessing the effects on the nervous system broadly. Zebrafish provide a unique opportunity to illuminate the genes of the druggable genome. Over the past five years, new tools have made it possible to make targeted changes in the zebrafish genome. Simultaneously, twelve new behavioral assays have been developed that can assess broadly the effects of genes or small molecules on the nervous system. This project combines these novel techniques for genome manipulation with high-throughput behavioral profiling to characterize genes of the druggable genome. Genes of interest will be knocked out in zebrafish using CRISPR-Cas systems, and the mutant animals will be subjected to a comprehensive panel of behavioral assays to assess effects on the nervous system. In parallel experiments, the same genes will be replaced in the zebrafish genome by a novel visualization/ablation cassette, enabling detailed 4D visualization of the in vivo expression patterns. Finally, a unique computational resource will enable facile identification of candidate ligands (probes) for the proteins of interest. These proof- of-concept experiments will focus on 15 genes selected to include both well- and poorly-characterized examples and will pave the way for a more comprehensive exploration of the druggable genome. Aims include: Aim 1. To characterize the nervous system functions of genes in the druggable genome. Aim 2. To develop tools for visualizing and manipulating the genes of the druggable genome. Aim 3. To identify candidate ligands for genes in the druggable genome. By providing detailed knock-out phenotyping, in vivo expression patterns, and a small molecule probe for each of the genes of interest, we hope to advance the important goal of illuminating the genes of the druggable genome.