A Highthroughput Targeted Genetic Screen for Modulators of Nociception

在我们的感官中，伤害感受，即检测有害刺激的能力，是生物体生存所必需的。 伤害感受诱导疼痛的感觉并促使避免疼痛源以使伤害最小化。 伤害性刺激由小直径初级外周神经元（伤害感受器）经由神经末梢检测， 投射到头部和身体的皮肤上，然后这些信息被传输到大脑，导致 对疼痛的知觉使人衰弱的慢性疼痛状况影响数亿人， 对个人和整个社会造成严重的身体、情感和经济负担。尽管 虽然有很大的进步，但关于疼痛刺激是如何被大脑感知和编码的，还有很多东西有待理解。 神经系统这导致缺乏有效的治疗方法和方法来识别有反应的患者 目前的治疗。此外，目前可用的基于药物的疗法具有许多有害的副作用， 影响和/或潜在的滥用和成瘾，而不是有效的治疗慢性 条件因此，更全面地了解这些感觉的生物学是至关重要的， 这可能会导致慢性疼痛的靶向治疗的发展。全转录组测序 已经提供了大量的关于基因的信息，这些基因优先在伤害感受神经元中表达 然而，还没有实用或有效的方法来研究这些基因在伤害感受中的作用， 传统的方法是缓慢的、麻烦的和昂贵的。在这里，我们建议使用相对 基于高通量CRISPR的基因组编辑策略，以视觉和行为方式探测 伤害感受器富集基因，利用斑马鱼模型系统。斑马鱼提供了一个有趣的模型 系统来研究伤害感受。斑马鱼幼鱼的伤害感受神经回路是高度相似的 与啮齿动物和人类等高等脊椎动物中的相似。此外，我们已经证明， 幼虫具有功能多样的外周和中枢神经系统， 刺激。此外，斑马鱼可以以低成本大量生产，并且它们的小尺寸允许 使用现有的高通量平台进行快速升级，这在其他脊椎动物系统中是不可能的 例如啮齿动物。使用CRISPR敲除/下调基因表达，我们的策略使我们能够评估 每周4个基因。基因敲除将发生在特异性标记 伤害感受器群体允许视觉评估任何给定基因在发育中的作用， 针对这些神经元。然后，我们将使用一个强大的幼虫运动行为测定来表征 基因突变对触觉、热、冷和化学刺激物的伤害性反应的影响。我们的初步 数据表明，我们可以识别影响不同疼痛模式的突变。我们认为这种基因 屏幕将提供一个资源，为社会感兴趣的发展神经电路和 疼痛的感知，并可能提供用于使人衰弱的疼痛病症的潜在疗法的靶点。

Among our senses nociception, the ability to detect noxious stimuli, is required for an organism's survival. Nociception induces the sensation of pain and prompts avoidance of the pain source so as to minimize injury. Noxious stimuli are detected by small diameter primary peripheral neurons (nociceptors) via nerve endings that project to the skin of the head and body, and this information is then transmitted to the brain, resulting in the conscious perception of pain. Debilitating chronic pain conditions affect hundreds of millions of people and impose a severe physical, emotional and economic burden on both individuals and society as a whole. Despite great advances, much remains to be understood about how painful stimuli are perceived and coded by the nervous system. This has resulted in a lack of effective therapies and methods to identify patients that respond to current treatments. Furthermore, currently available drug-based therapies have numerous deleterious side effects and/or potential for abuse and addiction, while not being effective for the treatment of chronic conditions. It is therefore vital to gain a more comprehensive understanding of the biology of these sensations, which could lead to the development of targeted treatment of chronic pain. Whole transcriptome sequencing has provided vast amounts of information about genes that are preferentially expressed in nociceptive neurons yet there have no practical or efficient methodologies to interrogate the role these genes play in nociception, as traditional approaches are slow, cumbersome and prohibitively expensive. Here we propose to use a relatively highthroughput CRISPR based genome editing strategy to visually and behaviorally probe the function of nociceptor enriched genes, utilizing the zebrafish model system. The zebrafish provides an intriguing model system to study nociception. The neural circuits underling nociception in zebrafish larvae are highly analogous to those found in higher vertebrates such as rodents and humans. Furthermore we've shown that zebrafish larvae have a functionally diverse peripheral and central nervous system and respond robustly to noxious stimuli. Additionally zebrafish can be generated in large numbers at low costs and their small size allows for rapid upscaling using existing high throughput platforms, which is not possible with other vertebrate systems such as rodents. Using CRISPR to knock out/down gene expression, our strategy allows us to assess the function of 4 genes per week. Gene knock out will occur in transgenic reporter lines that specifically label nociceptor populations allowing visual assessment of the role of any given gene in the development and targeting of these neurons. We will then use a robust larval locomotor behavioral assay to characterize the effects of genetic mutations on nociceptive response to touch, heat, cold and chemical irritants. Our preliminary data demonstrate that we can identify mutations affecting distinct pain modalities. We expect that this genetic screen will provide a resource for the community interested in the development of neural circuits and the perception of pain, and may provide targets for potential therapies for debilitating painful conditions.