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

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.