Site-Specific Gene Targeting Using Transcription Activator-Like Effector （TALE）-Based Nuclease in Brassica oleracea

• Published in: 植物学报：英文版 2013 (11), p.1092-1103
• Main Author: Zijian Sun Nianzu Li Guodong Huang Junqiang Xu Yu Pan Zhimin Wang Qinglin Tang Ming Song Xiaojia Wang
• Format: Article
• Language: English
• Subjects: DNA结合结构域; 基因打靶; 子样; 核酸酶; 甘蓝; 站点; 转录活化; 非同源末端连接
• ISSN: 1672-9072; 1744-7909

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.