A simple, flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system
2016
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
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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