J Integr Plant Biol. ›› 2016, Vol. 58 ›› Issue (8): 705-712.DOI: 10.1111/jipb.12474

Special Issue: CRISPR

• New Technology • Previous Articles     Next Articles

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

Hyeran Kim1†, Sang-Tae Kim1†, Jahee Ryu1, Min Kyung Choi1, Jiyeon Kweon2, Beum-Chang Kang1, Hyo-Min Ahn1, Suji Bae1, Jungeun Kim1,2, Jin-Soo Kim1,2,* and Sang-Gyu Kim1,*   

  1. 1Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea
    2Department of Chemistry, Seoul National University, Seoul, 08826, South Korea
  • Received:2015-12-23 Accepted:2016-03-03 Published:2016-03-04
  • About author:These authors contributed equally to this work.
    **Crrespondences: E-mail: jskim01@snu.ac.kr;sgkim@ibs.re.kr. (Kim is fully responsible for distributions of all materials associated with this article)

Abstract:

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.

Key words: AarI-mediated sgRNA cloning, CRISPR-Cas9 T-DNA binary vector, Exchangeable U6/U3 promoter, Gateway compatible Cas9 cloning

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