ULtiMATE system for rapid assembly of customized TAL effectors.
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.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Junjiao
J
Yang
Pengfei
P
Yuan
Dingqiao
D
Wen
Ying
Y
Sheng
Shiyou
S
Zhu
Yuezhou
Y
Yu
Xiang
X
Gao
Wensheng
W
Wei
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D015534:Trans-Activators; D004720:Endonucleases