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

作     者：Hyeran Kim Sang-Tae Kim Jahee Ryu Min Kyung Choi Jiyeon Kweon Beum-Chang Kang Hyo-Min Ahn Suji Bae Jungeun Kim Jin-Soo Kim Sang-Gyu Kim 

作者机构：Center for Genome Engineering Institute for Basic Science Department of Chemistry Seoul National University 

出 版 物：《Journal of Integrative Plant Biology》 (植物学报（英文版）)

年 卷 期：2016年第58卷第8期

页      面：705-712页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0703[理学-化学] 0902[农学-园艺学] 090102[农学-作物遗传育种] 

基　　金：supported by Institute for Basic Science (IBS-R021-D1) 

主　　题：Aar I mediated sg RNA cloning CRISPR Cas9 T DNA binary vector Exchangeable U6/U3 promoter Gateway compatible Cas9 cloning 

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.