CRISPR/Cas9‐mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis

作者：Chengzhou Mao, Li Zheng, Yimin Zhou, Haiyan Wu, Jingbo Xia, Chi‐Qian Liang, Xiaofang Guo, Wentao Peng, Hui Zhao, Weibin Cai, Soo‐Ki Kim, Kyu‐Sang Park, Dongqing Cai, Xufeng Qi · 发表于：The FASEB Journal · 年份：2018 · DOI：10.1096/fj.201800093 · 被引用次数：9 · 研究领域：CRISPR and Genetic Engineering、Pluripotent Stem Cells Research、Genetics, Aging, and Longevity in Model Organisms

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Z...