斑马鱼心脏发育候选基因Titin-Cap的CRISPR/Cas9打靶有效性分析  

Targeting Effectiveness Analysis of Heart Developmental Candidate Gene Titin-Cap with Gene Knockout Method CRISPR/Cas9

作　　者：袁月亲 罗世锋 颜尔聪 尹丽阳 漆轲婧 彭亚欣 万永奇 吴秀山 李永青[1] YUAN Yueqin;LUO Shifeng;YAN Ercong;YIN Liyang;QI Kejing;PENG Yaxin;WAN Yongqi;WU Xiushan;LI Yongqing(State Key Laboratory of Developmental Biology of Freshwater Fish,Hunan Normal University,Changsha 410081,Hunan,China)

机构地区：[1]心脏发育研究中心,省部共建淡水鱼类发育生物学国家重点实验室,教育部重点实验室,生命科学学院,湖南师范大学,湖南长沙410081

出　　处：《激光生物学报》2017年第5期458-465,457,共9页Acta Laser Biology Sinica

基　　金：国家自然科学基金面上项目(编号81470377);湖南省生物发育工程及新产品研发协同创新中心(2013-448-6)

摘　　要：CRISPR/Cas9基因打靶技术是近几年发展起来的一种高效率的定向打靶技术,被认为是遗传领域的革命性技术。Titin-Cap基因是本实验室已初步鉴定的斑马鱼心脏发育候选基因,且国内外目前尚无斑马鱼Titin-Cap基因的敲除品系。为了研究Titin-Cap基因在心脏发育过程中的作用机制,我们利用CRISPR/Cas9基因打靶技术建立斑马鱼Titin-Cap基因的敲除品系。测序结果显示,注射了CRISPR/Cas9 gRNA的胚胎出现双峰,说明在打靶位点附近出现了碱基缺失或插入,证明我们设计的gRNA是有效的。对F0代突变体成鱼的筛选中,测序结果同样显示有阳性结果。这些结果说明用CRISPR/Cas9基因打靶技术成功敲除了斑马鱼Titin-Cap基因,获得了Titin-Cap基因敲除的嵌合体斑马鱼。CRISPR/Cas9 technology,developed rapidly in recent years with a high targeting efficiency,is considered a revolutionary field of genetic technology.Titin-Cap gene is a candidate gene for zebrafish cardiac development,and there is no knockout strain of zebra fish Titin-Cap gene in the world so far.In order to study the mechanism of the Titin-Cap gene in heart development,here the CRISPR/Cas9 gene targeting technique was used to establish the Titin-Cap gene knockout strain in zebrafish.The sequencing results showed that the embryos injected with CRISPR/Cas9 gRNAs had bimodals,indicating that there was a base deletion or insertion near the target site.The sequencing results demonstrated that the gRNA was effective.In the screened F0 mutants,the sequencing results also showed positive results.All these results indicated that the Titan-Cap gene of the zebrafish was successfully knocked out with the CRISPR/Cas9 gene targeting technique,and the zebra fish of the Titin-Cap gene knockout was obtained.

关 键 词：心脏发育 斑马鱼 CRISPR/Cas9 Titin-Cap基因 

分 类 号：Q78[生物学—分子生物学]