title: A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.
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 Gateway(TM) 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.
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Hyeran
authors.initials: H
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Sang-Tae
authors.initials: ST
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Jahee
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Min Kyung
authors.initials: MK
authors.lastname: Choi
authors.affiliation: Department of Chemistry, Seoul National University, Seoul, 08826, South Korea.
authors.forename: Jiyeon
authors.initials: J
authors.lastname: Kweon
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Beum-Chang
authors.initials: BC
authors.lastname: Kang
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Hyo-Min
authors.initials: HM
authors.lastname: Ahn
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Suji
authors.initials: S
authors.lastname: Bae
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.|Department of Chemistry, Seoul National University, Seoul, 08826, South Korea.
authors.forename: Jungeun
authors.initials: J
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.|Department of Chemistry, Seoul National University, Seoul, 08826, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Yuseong-gu, Daejeon, 34047, South Korea.
authors.forename: Sang-Gyu
authors.initials: SG
authors.lastname: Kim
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D000072669:Gene Editing / Q000379:methods*; D017343:Genes, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D030821:Plants, Genetically Modified; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: AarI-mediated sgRNA cloning; CRISPR-Cas9 T-DNA binary vector; Exchangeable U6/U3 promoter; Gateway compatible Cas9 cloning