Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleav...Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.展开更多
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 consist...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.展开更多
综述了类转录激活因子效应物(TALEs)技术的研究进展及生物技术应用前景,重点介绍TALEs的结构特征、作用机理、广谱抗性基因的人工构建及利用类转录激活因子效应物核酸酶(Transcription activator like effector nucleases,TALENs)进行...综述了类转录激活因子效应物(TALEs)技术的研究进展及生物技术应用前景,重点介绍TALEs的结构特征、作用机理、广谱抗性基因的人工构建及利用类转录激活因子效应物核酸酶(Transcription activator like effector nucleases,TALENs)进行基因组定点修饰的策略。展开更多
基金Work on this topic in the authors’laboratories is supported by grants from:the Strategic Priority Research Program of the Chinese Academy of Sciences(number XDA01020106)the Ministry of Science and Technology of China 973 program(2011CB965200)+2 种基金the National Natural Science Foundation of China(81261130317)to MAEthe Bureau of Science,Technology and Information of Guangzhou Municipality(2012 J5100040)to MAE and JFgrants 2010U1-E00811-5 and ZNGI-2011-010 from the Guangzhou Municipality and the Chinese Academy of Sciences,respectively,to LL.
文摘Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.
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)
文摘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.
文摘综述了类转录激活因子效应物(TALEs)技术的研究进展及生物技术应用前景,重点介绍TALEs的结构特征、作用机理、广谱抗性基因的人工构建及利用类转录激活因子效应物核酸酶(Transcription activator like effector nucleases,TALENs)进行基因组定点修饰的策略。
