【摘要】 MYO7A是人类Usher综合征(US)的致病基因。由MYO7A突变导致的Usher综合征病例占Usher综合征1型病例的29%～55%。研究发现人类MYO7A突变能够导致Usher综合征1B型(USH1B),包括感觉神经性听力损伤及年龄依赖性视网膜色素变性(RP),但其分子机制尚不清楚。为了研究该基因在内耳及视网膜发育中的具体分子作用机制,利用Cloning free CRISPR/Cas9基因编辑技术构建斑马鱼myo7ab基因敲除品系。首先,通过分析软件筛选出该基因的敲除位点,利用聚合酶链式反应(PCR)技术扩增该基因的向导DNA(gDNA),再以gDNA为模板转录得到向导RNA(gRNA),将gRNA和Cas9蛋白共同注射到斑马鱼1细胞期胚胎中;随后,进行基因编辑的有效性检测。研究结果表明,CRISPR/Cas9系统对该基因的敲除有效。对其进行进一步筛选,成功获得斑马鱼myo7ab基因敲除突变品系,这为研究该基因在内耳及视网膜发育过程中的作用奠定了基础。更多还原

【Abstract】 MYO7 A is the causative gene of human Usher syndrome(US). Usher syndrome cases caused by MYO7 A mutation account for 29% to 55% of Usher syndrome type 1 cases. It has been reported that MYO7 A mutations can cause Usher syndrome type 1 B(USH1 B), including sensorineural hearing impairment and age-dependent retinitis pigmentosa(RP), whereas the molecular mechanism is still unclear. In order to elucidate the molecular mechanism of this gene in inner ear and retina development, we used cloning free CRISPR/Cas9 gene editing technology to establish the zebrafish myo7 ab knockout lines. Firstly, two knockout sites of the gene were screened by bioinformatics analysis. Next, the template guide DNA of this gene were amplified by polymerase chain reaction(PCR). Then, the template guide DNA were transcribed into the guide RNA. Finally, the guide RNA and Cas9 protein were co-injected into the 1-cell stage of zebrafish embryos. After the effectiveness analysis, it was proved that the CRISPR/Cas9 is effective in knocking out myo7 ab gene. The myo7 ab knockout line of zebrafish was established successfully after screening. The establishment of this line laid a foundation for studying the role of myo7 ab in the development of the inner ear and retina.更多还原