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 TA...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 Tetl locus, and proved that all can specifically target Tetl 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.展开更多
基金supported by the National Natural Science Foundation of China (No. Y211291131)the "Strategic Priority Research Program" of the Chinese Academy of Sciences (No. XDA01040109)
文摘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 Tetl locus, and proved that all can specifically target Tetl 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.
