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.
Bibliographical noteFunding Information:
The authors thank Yong-Long Chen (Southern University of Science and Technology, Shenzhen, China) for technical help and plasmids supplying. This work was supported by grants from the National Key Research and Development Program of China (2016YFE0204700 and 2017YFA0103302), the Major Research Plan of the National Natural Science Foundation of China–Key Program (91649203), the National Natural Science Foundation of China (81570222, 81770240, and 81270183), the Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306011), the Guangdong Science and Technology Planning Project (2014A050503043 and 2016A020221034), the New Star of Pearl River on Science and Technology of Guangzhou (2014J2200002), the Top Young Talents of Guangdong Province Special Support Program (87315007), the Foundation for Distinguished Young Talents in Higher Education of Guangdong Province (2013LYM0026), the Fundamental Research Funds for the Central Universities (21617436), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2013-693). C.-Z.M. and L.Z. contributed as co-first authors on this work. The authors declare no conflicts of interest.
All Science Journal Classification (ASJC) codes
- Molecular Biology