title: Rapid and efficient assembly of transcription activator-like effector genes by USER cloning.
abstract: Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
authors: Song
authors: S
authors: Wang
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors: Wei
authors: W
authors: Li
authors: Nanjing Agricultural University, Nanjing 210095, China.
authors: Shuo
authors: S
authors: Wang
authors: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: byhu@ioz.ac.cn.
authors: Baoyang
authors: B
authors: Hu
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D018390:Gene Targeting; D056426:Genetic Loci; D057809:HEK293 Cells; D006801:Humans; D006899:Mixed Function Oxygenases; D015202:Protein Engineering; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D003850:Deoxyribonuclease I; D043224:Deoxyribonuclease (Pyrimidine Dimer); D051981:Uracil-DNA Glycosidase
keywords: Golden Gate cloning; TAL effector; TALENs; Tet1; USER