利用CRISPR/Cas9技术建立斑马鱼Asb11基因敲除品系

Establishing the Asb11 Knockout Lines of Zebrafish with CRISPR/Cas9 Targeting Technology

Asb11基因被报道与斑马鱼Notch信号的激活有关,本研究室过去的研究显示该基因在心肌和骨骼肌中特异性表达。因此推测Asb11基因可能是心脏发育相关候选基因。为了阐明Asb11基因在斑马鱼心脏发育过程中的作用,本文利用CRISPR/Cas9打靶技术构建敲除Asb11基因的斑马鱼品系。首先在线分析筛选出Asb11基因最适合的打靶位点,然后PCR扩增出Asb11基因gRNA的双链c DNA,再将Asb11基因的gRNA和Hcas9的mRNA共同注射到斑马鱼胚胎Ⅰ细胞期胚胎中。进行打靶的有效性检测,发现Asb11基因的一号外显子出现了碱基的缺失,表明CRISPR/Cas9系统对Asb11基因的敲除是有效的。对其F0代、F1代、F2代进行筛选,成功获得了Asb11基因敲除的斑马鱼品系,为探究Asb11在心脏发育中的作用奠定了基础。

Asbl 1 gene was reported that it was associated with activation of Notch signaling pathway-. The previous re- sults in our lab showed that the gene expressed specifically in cardiac musele and skeletal nmsele. Therefore, it was hy- pothesized that Asbl 1 gene might be a candidate gene related to heart development. In this study, CRISPR/Cas9 targe- ting technology was employed to knockout zebrafish Asbl 1 gene. Firstly, the most suitable target sites in Asbl 1 gene were screened out on websites. Secondly, double-stranded DNA of the Asbll gene targeting gRNA was amplified by PCR. Thirdly, the Asbl 1 gene gRNA and Heas9 mRNA were co-injected into zebrafish embryos at l-cell stage. The results of the effectiveness-test showed that bases were deleted in the exonl of the zebrafish Asbll gene, suggesting that the CRISPR/Cas9 system was effective for knockout of Asbl 1 gene. After screening in the F0, F1 and F2 generations, the lines of zebrafish with Asbl 1 gene knocked-out were established successfully. The work laid a foundation for exploring the roles of Asbl 1 in zebrafish heart development.