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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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Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.(A, D and G) Partial sequences of HBEGF, ANTXR1, and LRP1 genes in genome containing TALENs binding regions (overlined for TALENL and underlined for TALENR). Restriction enzyme cutting sites are highlighted in yellow. (B, E and H) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting ANTXR2 gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (http://rsbweb.nih.gov/ij/). (C, F and I) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.
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pone-0075649-g003: Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.(A, D and G) Partial sequences of HBEGF, ANTXR1, and LRP1 genes in genome containing TALENs binding regions (overlined for TALENL and underlined for TALENR). Restriction enzyme cutting sites are highlighted in yellow. (B, E and H) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting ANTXR2 gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (http://rsbweb.nih.gov/ij/). (C, F and I) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.

Mentions: We have made hundreds of TALENs constructs by ULtiMATE targeting a variety of human genes. Figure 3 illustrated the detailed analysis for gene knockout effect on three representative genes. The knockout efficiencies indicated by genome PCR and restriction enzyme digestion were 64.63% (57.23%), 55.60% (33.12%), and 77.32% (48.83%) in HeLa (HEK293T) cells for HBEGF, ANTXR1, and LRP1, respectively. Sequencing analysis of 4-7 randomly picked clones confirmed the frame shift-caused stop of gene expression by variable indels. Although the presence of a restriction site between two TALENs binding regions could help to confirm the occurrence of indels and determine the gene knockout efficiency, it is often problematic to include restriction enzyme cutting site(s) in TALEN targeting regions. Instead of conducting Surveyor Nuclease digestion [3], we found out that direct sequencing of the TALENs-targeting region of the pooled clones could also reveal such information if the indels frequency was over 15-20%. As shown in Figure S2, the appearance of the baseline noise in the four-color sequencing chromatogram indicated that the occurrence of indels, and the level of which correlated well with the efficiency of TALENs-mediated cleavage.


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)

Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.(A, D and G) Partial sequences of HBEGF, ANTXR1, and LRP1 genes in genome containing TALENs binding regions (overlined for TALENL and underlined for TALENR). Restriction enzyme cutting sites are highlighted in yellow. (B, E and H) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting ANTXR2 gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (http://rsbweb.nih.gov/ij/). (C, F and I) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0075649-g003: Examples of disruption of genes in human cell lines by ULtiMATE-engineered TALENs.(A, D and G) Partial sequences of HBEGF, ANTXR1, and LRP1 genes in genome containing TALENs binding regions (overlined for TALENL and underlined for TALENR). Restriction enzyme cutting sites are highlighted in yellow. (B, E and H) Measurement of indel rates in TALENs-treated HeLa and HEK293T cells by restriction enzyme digestion. The uncleaved bands indicate potential indels. Both wild type and cells treated by TALENs targeting ANTXR2 gene are used as controls. The percentage of uncleaved band (indicated by red arrow) was measured using ImageJ (http://rsbweb.nih.gov/ij/). (C, F and I) Sequencing analysis of mutated alleles from 4-6 randomly selected TALENs clones (in HeLa cells). The TALENs binding sites (underlined) and restriction enzyme cutting sites (in yellow) are highlighted. Dashes and red letters indicate deletions and insertions, respectively.
Mentions: We have made hundreds of TALENs constructs by ULtiMATE targeting a variety of human genes. Figure 3 illustrated the detailed analysis for gene knockout effect on three representative genes. The knockout efficiencies indicated by genome PCR and restriction enzyme digestion were 64.63% (57.23%), 55.60% (33.12%), and 77.32% (48.83%) in HeLa (HEK293T) cells for HBEGF, ANTXR1, and LRP1, respectively. Sequencing analysis of 4-7 randomly picked clones confirmed the frame shift-caused stop of gene expression by variable indels. Although the presence of a restriction site between two TALENs binding regions could help to confirm the occurrence of indels and determine the gene knockout efficiency, it is often problematic to include restriction enzyme cutting site(s) in TALEN targeting regions. Instead of conducting Surveyor Nuclease digestion [3], we found out that direct sequencing of the TALENs-targeting region of the pooled clones could also reveal such information if the indels frequency was over 15-20%. As shown in Figure S2, the appearance of the baseline noise in the four-color sequencing chromatogram indicated that the occurrence of indels, and the level of which correlated well with the efficiency of TALENs-mediated cleavage.

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