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

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Title
A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system
Author(s)
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
Publication Date
2016-08
Journal
JOURNAL OF INTEGRATIVE PLANT BIOLOGY, v.58, no.8, pp.705 - 712
Publisher
WILEY-BLACKWELL
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. © 2016 Institute of Botany, Chinese Academy of Science
URI
https://pr.ibs.re.kr/handle/8788114/2793
ISSN
1672-9072
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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