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

作　　者：Zijian Sun Nianzu Li Guodong Huang Junqiang Xu Yu Pan Zhimin Wang Qinglin Tang Ming Song Xiaojia Wang 

机构地区：[1]Key Laboratory of Horticulture Science for Southern Mountainous Regions, Southwest University

出　　处：《Journal of Integrative Plant Biology》2013年第11期1092-1103,共12页植物学报（英文版）

基　　金：supported by grants from the National Basic Research Program of China (973 program, 2012CB113900);the National Natural Science Foundation of China (31071802);the Chongqing Natural Science Foundation (2011BA1002)

摘　　要：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.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.

关 键 词：Brassica oleracea double-strand break FRIGIDA gene targeting type III transcription activator-like effector-based nucleases non-homologous end-joining. 

分 类 号：Q78[生物学—分子生物学] Q754