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

Journal of integrative plant biology, 2016

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; (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 p...

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