CRISPR Screen for Hair Cell Development and Regeneration Regulators

项目总结 内耳的机械感觉毛细胞丢失是导致听力和平衡障碍的主要原因。 不幸的是，毛细胞丢失是永久性的，因为人类很少或根本没有能力再生恢复头发。 细胞。尽管最近在恢复哺乳动物毛细胞方面取得了进展，这是通过促进 在发育、功能恢复过程中使用的基因和途径的表达在很大程度上是不完整的 在有限的后天发育时间内。然而，促进再生的潜在新目标， 是有限的。我们建议对毛细胞转录调控因子进行高通量筛选。 斑马鱼侧线的发育和再生，这是一种易于获得的机械感觉系统 到活体动物的可视化和操控。虽然在组织方面肯定存在差异 侧线和内耳，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引导RNA来靶向特定的基因 并指示Cas9核酸酶进行突变破坏。它是高效的，允许这两个基因的修饰 在同一细胞内的等位基因，并导致遗传功能丧失。我们演示了我们可以使用CRISPR 以侧线基因表达为靶点。我们提出了一种系统的筛查方法来快速鉴定 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.