title: Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.
abstract: Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.
authors.affiliation: Key Laboratory of Horticulture Science for Southern Mountainous Regions, Southwest University, Chongqing, 400715, China.
authors.forename: Zijian
authors.initials: Z
authors.lastname: Sun
authors.forename: Nianzu
authors.initials: N
authors.lastname: Li
authors.forename: Guodong
authors.initials: G
authors.lastname: Huang
authors.forename: Junqiang
authors.initials: J
authors.lastname: Xu
authors.forename: Yu
authors.initials: Y
authors.lastname: Pan
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Wang
authors.forename: Qinglin
authors.initials: Q
authors.lastname: Tang
authors.forename: Ming
authors.initials: M
authors.lastname: Song
authors.forename: Xiaojia
authors.initials: X
authors.lastname: Wang
mesh_terms: D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001937:Brassica / Q000235:genetics*; D004252:DNA Mutational Analysis; D003851:Deoxyribonucleases / Q000378:metabolism*; D015870:Gene Expression; D018390:Gene Targeting; D017343:Genes, Plant / Q000235:genetics; D005820:Genetic Testing; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D015534:Trans-Activators; D003851:Deoxyribonucleases
keywords: Brassica oleracea; FRIGIDA; double-strand break; gene targeting; non-homologous end-joining; type III transcription activator-like effector-based nucleases