针对Fbxl5基因关键结构域F-box的Crispr/Cas9打靶有效性分析 | 激光生物学报 -- 中国光学期刊网

激光生物学报, 2016, 25 (6): 559, 网络出版: 2017-01-23

针对Fbxl5基因关键结构域F-box的Crispr/Cas9打靶有效性分析

The Analysis of Effectiveness of Crispr/Cas9 Targeting the Key Domain F-box of Fbxl5 Gene

论文大纲

高晶邓云陈宇高建芳傅雪珂尹丽阳万永奇吴秀山李永青 ^*

作者单位

湖南师范大学生命科学学院蛋白质化学及鱼类发育生物学教育部重点实验室心脏发育中心, 湖南长沙 410081

F-box基因家族基因敲除 Fbxl5 Fbxl5 F-box gene family CRISPR/Cas9 CRISPR/Cas9 gene mutation

摘要

为了在动物体内研究Fbxl5基因是通过什么机制导致斑马鱼心脏出现突变表型, 本文利用近几年兴起的CRISPR/Cas9打靶技术建立斑马鱼Fbxl5基因敲除品系。本文将打靶位点定位于Fbxl5的F-box结构域, 也就是Fbxl5的第五号外显子上。首先经过基因打靶网站分析筛选出针对Fbxl5基因F-box结构域最适合的打靶位点, 扩增出Fbxl5基因CRISPR/Cas9打靶双链DNA, 并转录为RNA, 与Hcas9共注射至斑马鱼胚胎。最后, 在注射48 h后对Fbxl5基因CRISPR/Cas9打靶的有效性进行检测。首先在注射48 h之后收集胚胎提取基因组DNA, 用特异性引物进行PCR扩增; 将纯化后的Fbxl5基因PCR产物连接到pMD18-T载体, 再经质粒提取, 测序分析, 通过与WT斑马鱼基因组序列进行比对发现Fbxl5-9号在PAM序列AGG下游缺失了4个碱基。证明该CRISPR/Cas9 系统在敲除心脏发育候选基因Fbxl5是有效的, 该研究为最终获得Fbxl5基因敲除斑马鱼奠定了良好的基础。

Abstract

Our previous results showed that disturbing expression of Fbxl5 resulted into heart defects in zebra fish embryos. In order to reveal a possible molecular mechanism in vivo underlying the effect of Fbxl5 gene, here we used CRISPR/Cas9 targeting technology which is very popular in recent years to establish the Fbxl5 knockout lines of zebra fish. The F-box structural domain was chosen as the target site. Firstly, through the website analysis, we identified the most appropriate target site targeted on Fbxl5 F-box domain. Secondly, double strand DNAs for Fbxl5 specific gRNA were synthesized by PCR and were transcript to mRNA. Lastly, Fbxl5 gRNA and capped Hcas9 mRNA were micro-injected into zebra fish embryos of one cell phase. After the microinjected, we detected the targeting effectiveness of zebra fish endogenous gene Fbxl5. The second target site in the fifth exon of gene Fbxl5, was found to produce a 4-bases deletion. This consequence indicated that CRISPR/Cas9 system was effective in knocking out gene Fbxl5 and it laid a good foundation for obtaining zebra fish lines knockouted Fbxl5 gene. In summary, the results in the present study lay the foundation for understanding the mechanism underlying Fbxl5 in heart development in vivo.

参考文献

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高晶, 邓云, 陈宇, 高建芳, 傅雪珂, 尹丽阳, 万永奇, 吴秀山, 李永青. 针对Fbxl5基因关键结构域F-box的Crispr/Cas9打靶有效性分析[J]. 激光生物学报, 2016, 25(6): 559. GAO Jing, DENG Yun, CHEN Yu, GAO Jianfang, FU Xueke, YIN Liyang, WAN Yongqi, WU Xiushan, LI Yongqing. The Analysis of Effectiveness of Crispr/Cas9 Targeting the Key Domain F-box of Fbxl5 Gene[J]. Acta Laser Biology Sinica, 2016, 25(6): 559.

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