CRISPR-dCpf1精细调控Wnt、Notch、TGF-β、Atoh1基因群促进前庭毛细胞再生的研究

通过Atoh1基因过表达诱导支持细胞向毛细胞分化是内耳毛细胞再生领域的重要进展，但仍存在支持细胞数量不足，新生毛细胞尚不成熟等问题，需对Atoh1介导的细胞转化过程加以精细调控。利用RNA-Seq技术和生物信息学分析发现Wnt、Notch、TGF-β三个信号通路在毛细胞再生过程中发挥重要作用，申请人前期开发的工程化改造的CRISPR-dCpf1技术可实现多基因精细化转录调控。因此推测：利用工程化改造的CRISPR-dCpf1技术，在过表达Atoh1同时精确调控Wnt、Notch、TGF-β将有效促进内耳毛细胞功能性再生。为了验证该假说，本课题以前庭毛细胞为研究对象，建立椭圆囊斑体外损伤培养模型和前庭功能损伤动物模型，采用工程化改造的CRISPR-dCpf1技术在体外和体内分别调控Atoh1、Wnt、Notch、TGF-β相关基因转录，探索内耳毛细胞再生新策略。

Atoh1 expression induced hair-cell regeneration is important progress, but there are still many problems need to be solved. This suggests that the cell transformation process mediated by Atoh1 should be carefully regulated. Using RNA-Seq technology and bioinformatics analysis, the applicant found that Wnt, Notch and TGF-β signaling pathways play important roles in the process of hair cell regeneration. The engineered CRISPR-dCpf1 technology developed by the applicant could complete the fine transcriptional regulation of multiple genes. Therefore, it was speculated that using the engineered CRISPR-dCpf1 technology to accurately regulate Wnt, Notch and TGF-β while overexpressing Atoh1 would effectively promote functional regeneration of hair cells. In order to test this hypothesis, this project took vestibular hair cells as the research object, established in vitro injury utricle culture model and vestibular function injury animal model, and used the engineered crispr-dcpf1 technology to regulate the transcription of Atoh1, Wnt, Notch and TGF-β related gene groups in vitro and in vivo, respectively. These studies will explore new technique and strategy for regeneration of hair cells in the inner ear.