TALEN Plasmids and Kits

The plasmids pC-GoldyTALEN and RCIsricpt-GoldyTALEN are designed as destination vectors for the Voytas lab Golden Gate TALEN kit. The GoldyTALEN scaffold is truncated at both the N and C terminus and induces mutation at rates much higher than the parental vectors. pC-GoldyTALEN directs expression of TALENs from a truncated CAGs promoter. RCIscript-GoldyTALEN is designed for in vitro synthesis of TALEN mRNAs. Both 5’ and 3’ Xenopus β-globin UTRs are included in the vector to enhance expression of the message. Both vectors utilize homodimeric FokI domains.

Plasmids pTAL5-BB and pTAL6-BB are designed as alternative destination vectors for the Golden Gate TALEN kit (Bogdanove/Voytas) in order to generate TALORs (TAL Orthongal Repressors) that can be used to custom repress gene expression in yeast. TALORs consist of the DNA-binding domain of a TALE, along with strong nuclear localisation tags and repress transcription initiation when targeted to DNA sequences within core promoter regions. pTAL5-BB contains the GAL1 promoter, placing TALORs built into this vector under galactose-inducible expression. pTAL6-BB contains the TEF1 promoter, giving constitutive expression of TALORs built into this vector.

pCS2TAL3-DD and pCS2TALE3-RR are next generation TALEN backbone vectors designed for use with the Voytas Golden Gate TALEN Kit and are used in place of pTAL1, 2, 3, or 4. For both plasmids sequence positions 1214–2210 of pTAL3 were cloned into a pCS2 expression vector resulting in shorter N- and C-terminal tal protein segments (136AA and 63AA, respectfully). This next generation architecture has been shown to increase mutation induction when using TALENs. The FokI domains (DD, RR) used are obligate heterodimers that require cloning of left and right TALEN monomer proteins into opposite vectors.

pCAG-T7-TALEN(Sangamo)-Destination constructs were designed for optimal mammalian expression of Voytas Golden Gate-assembled TALEN, both in microinjected embryos and transfected cells. TALEN expression is driven by the strong CAG promoter or can be achieved by in vitro mRNA synthesis from the T7 promoter. Truncations were introduced to the N- and C-terminus of the pTAL3 TALEN backbone, which were initially published by Sangamo BioSciences (N153AA, C63AA) and showed robust cleavage activity in several later studies. pCAG-T7-TALEN(Sangamo)-Destination vectors are available with homodimeric or enhanced heterodimeric (ELD, KKR mutations) FokI domains.

This accessory pack contains modified pFUS array vectors, destination vectors and a reporter vector for mammalian cell-based validation assay that are designed for use with the Golden Gate TALEN and TAL Effector Kit. These modified pFUS vectors can reduce the number of module plasmids and improve the success rate of Golden Gate assembly. Destination vectors, pcDNA-TAL-NC2 and pCAGGS-TAL-NC2, are mammalian expression/mRNA synthesis vectors containing unique shorter N- and C-terminal domains (N153AA, C47AA). The reporter vector pGL4-SSA can be used for single-strand annealing assay, which enables the evaluation of TALEN activities in cultured cells.

These new destination vectors can be used to create TALE-transcription factors and are used at the final assembly step in the Golden Gate protocol. Both plasmids 47388 and 47389 include a Flag epitope tag and an SV40 NLS at the N terminus, a 152-residue deletion from the N terminus of the wild-type TALE protein (previously shown to preserve the DNA-binding ability of TALEs), 63 wild-type TAL amino acids after the repeat domain, and C-terminal SV40 NLS and HA tags. Plasmid 47389 also contains the VP64 domain (four repeats of the minimal activation domain of VP16) at the C terminus.

These plasmids were designed for mammalian expression of fluorescent TAL effectors to visualize subcellular positioning of target DNA sequences in living cells (TALE-mediated Genome Visualization [TGV]). pTALYM3 and pTALYM4 contain TALE fused with mClover and mRuby2, respectively, and can be used as destination vectors for Voytas Golden Gate-assembly kit.

Application of TALEN technology in hPSCs

pTAL7a and pTAL7b vectors were designed for use with the Voytas Lab Golden Gate TALEN kit, replacing pTAL4. The pTAL7 destination plasmids were optimized for TALEN expression in mammalian systems including hard-to-transfect cell lines. Features include (i) Esp3I (BsmBI) restriction sites for full compatibility with the Golden Gate TALEN kit, (ii) a lacZ fragment for blue/white-screening in E.coli, (iii) CAG promoter and Kozak sequence to drive efficient TALEN expression in mammalian cells, (iv) an improved, truncated TALE backbone architecture as established by Miller et al. (PMID: 21179091), as well as (v) distinct selection cassettes on pTAL7a and pTAL7b for enrichment of double-transfected mammalian cells.

In addition, a T7 promoter was included for generating TALEN mRNAs through in vitro transcription as an alternative. Based on transient double-selection of transfected cells, the system has successfully been employed for deletion mutagenesis and gene targeting in human and mouse pluripotent stem cells.

The TALE plasmid vectors described on this page are designed to target and edit the epigenome of putative enhancers in mammalian cells. These plasmids allow for cloning DNA fragments encoding TAL effector repeat arrays (assembled using the REAL, REAL-Fast, or FLASH methods) for expression as TALE-LSD1 fusions.

It was recently shown that TALE fusions with the histone demethylase LSD1 (Lysine specific demethylase) made using these plasmids along with the FLASH method can decrease the amount of Histone H3 lysine 4 (H3K4) di-methylation at the target locus (Mendenhall et al., 2013). This epigenome editing was also shown to alter regulation of nearby gene expression.

The vectors listed below have the EF1α promoter for efficient mammalian cell expression, and encode one of three 0.5 TALE repeats (C, T, and G) and the full length human LSD1 at the carboxy-terminus of the protein.

pBlue-TAL was designed for use with the Voytas Lab Golden Gate TALEN kit. This backbone is suitable for the TALEN-based generation of germline mutations in Bombyx mori and Drosophila melanogaster.