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.
Highlights
Natural TAL effectors (TALEs), originally identified from bacteria Xanthomonas, mimic eukaryotic transcription factors to reprogram host cells [1]
The TAL effector-targeting domain has been shown to create site-specific DNA double-strand breaks (DSBs) when fused with the catalytic domain of the FokI nuclease [3,5,6,7]. Such TAL effector nucleases (TALENs) work in pairs each of which is designed to fuse the FokI monomer and binds opposing DNA target sites separated by an appropriative spacer
The DNA fragments applicable to uracil-specific excision reagent (USER) fusion were exclusively obtained from PCR reactions using special uracil-containing primers, which were performed with polymerases that could incorporate a deoxyadenine opposite a dU, such as PfuTurbo Cx Hotstart DNA polymerase
Summary
Natural TAL effectors (TALEs), originally identified from bacteria Xanthomonas, mimic eukaryotic transcription factors to reprogram host cells [1]. A typical TALE contains a central DNA-binding region of tandem repeats of 34 amino acids, with each repeat targeting a nucleotide using repeat variable diresidues (RVDs) at positions 12 and 13 [1,2]. This modular DNA-binding feature allows protein engineering by design-based assembly of TALE repeats for use in gene targeting [3,4]. The TAL effector-targeting domain has been shown to create site-specific DNA double-strand breaks (DSBs) when fused with the catalytic domain of the FokI nuclease [3,5,6,7]. The USERTM enzyme mix, a mixture of glycosidase (UDG) and DNA glycosylase-lyase endo VIII, was used to remove the dU residues to generate 3’-protruding sticky ends in PCR products [20]
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