Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

文献类型： 外文期刊

第一作者： Wang, Xianlong

作者： Wang, Xianlong;Cao, Chunwei;Huang, Jiaojiao;Hai, Tang;Zheng, Qiantao;Zhang, Hongyong;Qin, Guosong;Miao, Xiangnan;Wang, Hongmei;Zhou, Qi;Zhao, Jianguo;Zhou, Jinwei;Cao, Suizhong;Huang, Jiaojiao;Zheng, Qiantao;Zhang, Hongyong;Wang, Hongmei;Zhou, Qi;Zhao, Jianguo;Wang, Yanfang

作者机构：

期刊名称：SCIENTIFIC REPORTS （ 影响因子：4.379； 五年影响因子：5.133 ）

ISSN： 2045-2322

年卷期： 2015 年 5 卷

页码：

收录情况： SCI

摘要： Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序