A Highthroughput Targeted Genetic Screen for Modulators of Nociception

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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.