A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

查看全文 作　　者：Hyeran [1]Kim;Sang-Tae [1]Kim;Jahee [1]Ryu;Min Kyung [1]Choi;Jiyeon [2]Kweon;Beum-Chang [1]Kang;Hyo-Min [1]Ahn;Suji [1]Bae;Jungeun [1,2]Kim;Jin-Soo [1,2]Kim;Sang-Gyu [1]Kim

机构地区：[1]Center for Genome Engineering, Institute for Basic Science;[2]Department of Chemistry, Seoul National University

出　　处：《Journal of Integrative Plant Biology》索引2016年第58卷第8期,共8页

基　　金：supported by Institute for Basic Science (IBS-R021-D1) 摘　　要：CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes(Sp Cas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA(sg RNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures:(1) annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and(2) ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant. 关键词：Aar I-mediated sg RNA cloning CRISPR-Cas9 T-DNA binary vector Exchangeable U6/U3 promoter Gateway compatible Cas9 cloning

相关文献

参考文献（45）
引证文献（4）
耦合文献（0）

参考文献(45)

引证文献(4)

互联网出版许可证新出网证(渝)字10号全国400电话 - 免长途话费

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

参考文献(45)

引证文献(4)

用户提示