CRISPR Screen for Hair Cell Development and Regeneration Regulators

毛细胞发育和再生调节因子的 CRISPR 筛选

基本信息

批准号：
9186821
负责人：
David W Raible
金额：
$ 23.18万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2018-06-30
项目状态：
已结题

项目摘要

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.

项目摘要内耳的机械感力毛细胞损失是听力和平衡疾病的主要原因。不幸的是，毛细胞损失是永久的，因为人类几乎没有或没有再生恢复头发的能力细胞。尽管最近通过促进哺乳动物的毛细胞恢复了毛细胞的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不完整，并且在有限的产后发展窗口中。然而，潜在的促进再生的新目标有限。我们建议对毛细胞的转录调节器进行高通量屏幕斑马鱼侧线的开发和再生，这是一种容易访问的机械感觉系统在活体动物中可视化和操纵。虽然在组织中肯定存在差异横向线和内耳，对毛细胞中使用的许多基因都有基本的保护发展和功能。 CRISPR系统使用易于合成的指南RNA来靶向特定基因并直接CAS9核酸酶进行诱变断裂。它高效，可以修改两个基因等位基因在同一细胞内，导致功能的遗传丧失。我们证明我们可以使用CRISPR 靶向基因在横向线中的表达。我们提出了一个系统的屏幕，以快速识别调节毛细胞发育和再生的基因。