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A simple, flexible and high‐throughput cloning system for plant genome editing via CRISPR‐Cas system
Author(s) -
Kim Hyeran,
Kim SangTae,
Ryu Jahee,
Choi Min Kyung,
Kweon Jiyeon,
Kang BeumChang,
Ahn HyoMin,
Bae Suji,
Kim Jungeun,
Kim JinSoo,
Kim SangGyu
Publication year - 2016
Publication title -
journal of integrative plant biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.734
H-Index - 83
eISSN - 1744-7909
pISSN - 1672-9072
DOI - 10.1111/jipb.12474
Subject(s) - biology , cas9 , crispr , computational biology , guide rna , subgenomic mrna , agrobacterium , genetics , genome editing , expression vector , genome , oligonucleotide , restriction site , transformation (genetics) , dna , restriction enzyme , gene , recombinant dna
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 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 Gateway TM system 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.