Journal
PLOS ONE
Volume 8, Issue 9, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0075649
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Funding
- Major State Basic Research Development Program of China [2010CB911800]
- National Science Foundation of China [NSFC31070115, NSFC31170126]
- 985 Project of Peking University
- Peking-Tsinghua Center for Life Sciences
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Engineered TAL-effector nucleases (TALENs) and TALE-based constructs have become powerful tools for eukaryotic genome editing. Although many methods have been reported, it remains a challenge for the assembly of designer-based TALE repeats in a fast, precise and cost-effective manner. We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector) system for speedy and accurate assembly of customized TALE constructs. This method takes advantage of uracil-specific excision reagent (USER) to create multiple distinct sticky ends between any neighboring DNA fragments for specific ligation. With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation. This system has been demonstrated to produce both functional TALENs for effective gene knockout and TALE-mediated gene-specific transcription activation (TALE-TA). The feature of both ease-of-operation and high efficiency of ULtiMATE system makes it not only an ideal method for biologic labs, but also an approach well suited for large-scale assembly of TALENs and any other TALE-based constructions.
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