摘要: 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 GatewayTM 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.
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期刊:
JIPB
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分类:
生物学
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植物学
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植物学研究、实验与植物演化、发展
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引用:
ChinaXiv:201605.00420
(或此版本
ChinaXiv:201605.00420V1)
DOI:10.12074/201605.00420V1
CSTR:32003.36.ChinaXiv.201605.00420.V1
- 推荐引用方式:
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.A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.中国科学院科技论文预发布平台.[ChinaXiv:201605.00420V1]
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