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

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• 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 and Sang-Gyu Kim
• 刊名：Journal of Integrative Plant Biology
• 出版年：2016
• 出版时间：August 2016
• 年：2016
• 卷：58
• 期：8
• 页码：705-712
• 全文大小：1403K
• ISSN：1744-7909

文摘

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19−20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI 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 Gateway^TM system and unique EcoRI/XhoI 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.