Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.
Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.
复制标题
ROSA26靶向的绵羊基因通过CRISPR-CAS9系统敲入。
DOI：
10.1038/srep24360
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发表时间：
2016-04-11
影响因子：
4.6
通讯作者：
Wang C
中科院分区：
文献类型：
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作者：
Wu M;Wei C;Lian Z;Liu R;Zhu C;Wang H;Cao J;Shen Y;Zhao F;Zhang L;Mu Z;Wang Y;Wang X;Du L;Wang C
我们最近在为所需序列设计具有特异性的DNA结合因子方面的进展导致了基因工程的一场革命，使对基因组进行定向改变变得相对容易。促进特定和精确的基因组编辑的技术，如敲入，对于确定基因的功能和了解基本的生物过程至关重要。CRISPR/Cas9系统最近成为哺乳动物功能基因组研究的有力工具。Rosa26基因在几乎所有组织中都能编码一种非必需的核RNA，成为外源基因插入的热点。在这里，我们描述了高效、精确的CRISPR/Cas9介导的整合，使用的是以绵羊基因组rosa26为靶点的tGFP序列供体载体。我们成功地将外源tGFP(TurboGFP)基因高效整合到FRAME中。由于其简单、设计灵活、效率高，我们建议CRISPR/Cas9介导的敲入将成为一种标准的转基因绵羊世代方法。
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.