CRISPR Screen for Hair Cell Development and Regeneration Regulators

项目摘要 内耳中的机械感觉毛细胞损失是听力和平衡障碍的主要原因。 不幸的是，头发细胞的损失是永久性的，因为人类几乎没有或没有再生恢复头发的能力。 细胞虽然最近在哺乳动物毛细胞的恢复方面取得了进展， 发育过程中使用的基因和途径的表达，功能恢复在很大程度上是不完整的， 在产后发育的有限窗口内。然而，促进再生的潜在新目标， 是有限的。我们建议进行高通量筛选毛细胞转录调控因子 斑马鱼侧线的发育和再生，这是一个很容易接近的机械感觉系统 to visualization可视化and manipulation操纵in living活的animals动物.虽然在组织上确实存在差异， 在侧线和内耳中，毛细胞中使用的许多基因基本上是保守的。 发展和功能。CRISPR系统使用容易合成的指导RNA来靶向特定基因 并指导Cas9核酸酶进行诱变断裂。它是高效的，允许修饰两个基因 在同一个细胞内的等位基因，并导致遗传功能的丧失。我们证明了我们可以使用CRISPR 来靶向侧线基因的表达。我们提出了一个系统的屏幕快速识别 调节毛细胞发育和再生的基因。

PROJECT SUMMARY Mechanosensory hair cell loss in the inner ear is a leading cause of hearing and balance disorders. Unfortunately hair cell loss is permanent as humans have little or no capacity for regenerative recovery of hair cells. Although there has been recent progress in the restoration of hair cells in mammals by promoting expression of genes and pathways used during development, functional restoration is largely incomplete and within a limited window of postnatal development. Potential new targets for promoting regeneration, however, are limited. We propose to undertake a high-throughput screen for transcriptional regulators of hair cell development and regeneration in the zebrafish lateral line, a mechanosensory system that is readily accessible to visualization and manipulation in living animals. While there are certainly differences in the organization of the lateral line and inner ear, there is fundamental conservation of many of the genes used in hair cell development and function. The CRISPR system uses easily synthesized guide RNAs to target specific genes and direct Cas9 nuclease to make mutagenic breaks. It is highly efficient, allowing modification of both gene alleles within the same cell and resulting in genetic loss of function. We demonstrate that we can use CRISPR to target expression of genes in the lateral line. We propose a systematic screen for rapid identification of genes that regulate hair cell development and regeneration.