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PLoS One. 2013 Sep 27;8(9):e75649. doi: 10.1371/journal.pone.0075649. eCollection 2013.

ULtiMATE system for rapid assembly of customized TAL effectors.

Author information

1
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.

Abstract

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.

PMID:
24228087
PMCID:
PMC3815405
DOI:
10.1371/journal.pone.0075649
[Indexed for MEDLINE]
Free PMC Article

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