title: A simple and efficient method for assembling TALE protein based on plasmid library.
abstract: DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.
authors.affiliation: College of Animal Science and Technology, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaan'xi, PR China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Zhang
authors.forename: Duo
authors.initials: D
authors.lastname: Li
authors.forename: Huarong
authors.initials: H
authors.lastname: Xu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xin
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.forename: Lixia
authors.initials: L
authors.lastname: Ma
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Zhilong
authors.initials: Z
authors.lastname: Chen
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D015723:Gene Library; D000090062:Kruppel-Like Factor 4; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D014974:Xanthomonas
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4