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

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 b...

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Bibliographic Details
Published in:Journal of integrative plant biology 2016-08, Vol.58 (8), p.705-712
Main Authors: Kim, Hyeran, Kim, Sang-Tae, Ryu, Jahee, Choi, Min Kyung, Kweon, Jiyeon, Kang, Beum-Chang, Ahn, Hyo-Min, Bae, Suji, Kim, Jungeun, Kim, Jin-Soo, Kim, Sang-Gyu
Format: Article
Language:English
Subjects:
AarI-mediated sgRNA cloning
Arabidopsis - genetics
Base Sequence
Cloning, Molecular - methods
CRISPR-Cas Systems - genetics
CRISPR-Cas9 T-DNA binary vector
Exchangeable U6/U3 promoter
Gateway compatible Cas9 cloning
Gene Editing - methods
Genes, Plant
Genetic Vectors
Genome, Plant
Plants, Genetically Modified
RNA, Guide, CRISPR-Cas Systems - genetics
Transformation, Genetic
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Summary: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.
ISSN:1672-9072
1744-7909
DOI:10.1111/jipb.12474