斑马鱼myo7ab基因敲除品系的构建

The Establishment of the Zebrafish myo7ab Knockout Lines

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。

MYO7A is the causative gene of human Usher syndrome(US).Usher syndrome cases caused by MYO7A muta-tion account for 29%to 55%of Usher syndrome type 1 cases.It has been reported that MYO7A mutations can cause Usher syn-drome type 1B(USH1B),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 devel-opment,we used cloning free CRISPR/Cas9 gene editing technology to establish the zebrafish myo7ab knockout lines.Firstly,two knockout sites of the gene were screened by bioinformatics analysis.Next,the template guide DNA of this gene were ampli-fied 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 myo7ab gene. The myo7ab knockout line of zebrafish was established suc- cessfully after screening. The establishment of this line laid a foundation for studying the role of myo7ab in the development of the inner ear and retina.