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ULtiMATE system for rapid assembly of customized TAL effectors.

Yang J, Yuan P, Wen D, Sheng Y, Zhu S, Yu Y, Gao X, Wei W - PLoS ONE (2013)

Bottom Line: Engineered TAL-effector nucleases (TALENs) and TALE-based constructs have become powerful tools for eukaryotic genome editing.We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector) system for speedy and accurate assembly of customized TALE constructs.With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Effects of TALE-TAs in human cell lines.(A) Design and structure of a representative luciferase reporter, Triple A-Luc, and its corresponding TALE transactivator, TALE-(NI)7,8,9. The reporter contains an artificial TALE-binding sequence (CTGGCCAAATACGTA) upstream a mini CMV promoter, followed by the Firefly luciferase gene. TALE-(NI)7,8,9 carries the Triple A binding TALE, fused with VP64 viral activation domain. The TALE-binding sequences for Triple-A, -C, -G and -T reporters differ only in 3 consecutive bases in the middle. (B) The binding activity of each TALE-TAs is determined by measuring the relative luciferase units (RLU) for their corresponding reporter activity after normalization with a co-transfected Renilla expressing vector pRL-TK. Error bars indicate standard deviations of four replicates.
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pone-0075649-g002: Effects of TALE-TAs in human cell lines.(A) Design and structure of a representative luciferase reporter, Triple A-Luc, and its corresponding TALE transactivator, TALE-(NI)7,8,9. The reporter contains an artificial TALE-binding sequence (CTGGCCAAATACGTA) upstream a mini CMV promoter, followed by the Firefly luciferase gene. TALE-(NI)7,8,9 carries the Triple A binding TALE, fused with VP64 viral activation domain. The TALE-binding sequences for Triple-A, -C, -G and -T reporters differ only in 3 consecutive bases in the middle. (B) The binding activity of each TALE-TAs is determined by measuring the relative luciferase units (RLU) for their corresponding reporter activity after normalization with a co-transfected Renilla expressing vector pRL-TK. Error bars indicate standard deviations of four replicates.

Mentions: Concerned with the degenerate sequences used in the TALE units, we wanted to verify whether TALEs we produced have specific DNA binding capability. To test this, we constructed four luciferase reporters as well as their corresponding TALEs fused with VP64 (Figure 2). The reporters contained an artificial TALE-binding sequence upstream a mini CMV promoter, followed by firefly luciferase gene. The TALE-binding sequence for Triple-A reporter contains 3 consecutive A (i.e., CTGGCCAAATACGTA) from position 7 to 9 (Figure 2A). Likewise, Triple-C, -G and -T reporters differ only in these 3 consecutive bases. Co-introduced into cells, TALE-


ULtiMATE system for rapid assembly of customized TAL effectors.

Yang J, Yuan P, Wen D, Sheng Y, Zhu S, Yu Y, Gao X, Wei W - PLoS ONE (2013)

Effects of TALE-TAs in human cell lines.(A) Design and structure of a representative luciferase reporter, Triple A-Luc, and its corresponding TALE transactivator, TALE-(NI)7,8,9. The reporter contains an artificial TALE-binding sequence (CTGGCCAAATACGTA) upstream a mini CMV promoter, followed by the Firefly luciferase gene. TALE-(NI)7,8,9 carries the Triple A binding TALE, fused with VP64 viral activation domain. The TALE-binding sequences for Triple-A, -C, -G and -T reporters differ only in 3 consecutive bases in the middle. (B) The binding activity of each TALE-TAs is determined by measuring the relative luciferase units (RLU) for their corresponding reporter activity after normalization with a co-transfected Renilla expressing vector pRL-TK. Error bars indicate standard deviations of four replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815405&req=5

pone-0075649-g002: Effects of TALE-TAs in human cell lines.(A) Design and structure of a representative luciferase reporter, Triple A-Luc, and its corresponding TALE transactivator, TALE-(NI)7,8,9. The reporter contains an artificial TALE-binding sequence (CTGGCCAAATACGTA) upstream a mini CMV promoter, followed by the Firefly luciferase gene. TALE-(NI)7,8,9 carries the Triple A binding TALE, fused with VP64 viral activation domain. The TALE-binding sequences for Triple-A, -C, -G and -T reporters differ only in 3 consecutive bases in the middle. (B) The binding activity of each TALE-TAs is determined by measuring the relative luciferase units (RLU) for their corresponding reporter activity after normalization with a co-transfected Renilla expressing vector pRL-TK. Error bars indicate standard deviations of four replicates.
Mentions: Concerned with the degenerate sequences used in the TALE units, we wanted to verify whether TALEs we produced have specific DNA binding capability. To test this, we constructed four luciferase reporters as well as their corresponding TALEs fused with VP64 (Figure 2). The reporters contained an artificial TALE-binding sequence upstream a mini CMV promoter, followed by firefly luciferase gene. The TALE-binding sequence for Triple-A reporter contains 3 consecutive A (i.e., CTGGCCAAATACGTA) from position 7 to 9 (Figure 2A). Likewise, Triple-C, -G and -T reporters differ only in these 3 consecutive bases. Co-introduced into cells, TALE-

Bottom Line: Engineered TAL-effector nucleases (TALENs) and TALE-based constructs have become powerful tools for eukaryotic genome editing.We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector) system for speedy and accurate assembly of customized TALE constructs.With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus