Transcription activator-like (TAL) effectors are DNA binding proteins produced by Xanthomonas bacteria when they infect plants. These proteins can activate the expression of plant genes by recognizing and binding host plant promoter sequences through a central repeat domain consisting of a variable number of ~34 amino acid repeats. The residues at the 12th and 13th positions of each repeat are hyper-variable. There appears to be a simple one-to-one code between these two critical amino acids in each repeat and each DNA base in the target sequence, e.g., NI = A, HD = C, NG = T, and NN = G or A.
TAL effectors have been utilized to create site-specific gene-editing tools by fusing target sequence-specific TAL effectors to nucleases (TALENs), transcription factors (TALE-TFs), and other functional domains. These fusion proteins can recognize and bind chromosome target sequences specifically to execute their gene editing functions, such as gene knockout, knockin (with donor plasmid), mutagenesis, activation, repression, and more. Unlike zinc fingers’ nucleotide triplet recognition, TAL effector domains recognize single nucleotides, which allows researchers to be able to specifically target whatever sequence they want.
Advantages
Target any sequence in any cell
Highly sequence-specific genome editing
For gene knockout, knockin, mutagenesis, activation, repression and more
Flexible TAL effector design of binding and functional domains, such as TALEN and TALE-TF
More potential off-target effects than TALENs and ZFNs
Multiplexing
Rarely used
Capable
References
Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009 326(5959):1509-12
Moscou, M. et al. A simple cipher governs DNA recognition by TAL effectors. Science. 2009 326(5959):1501
Christian, M. et al. Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. DOI: 10.1534/genetics.110.120717
Morbitzera, R. et al. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. www.pnas.org/cgi/doi/10.1073/pnas.1013133107
Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12 e82 doi:10.1093/nar/gkr218
Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Research, 2011, Vol. 39, No. 14 6315–6325 doi:10.1093/nar/gkr188
Zhang, F. et al. Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors. Nat Biotechnol. 2011 February ; 29(2): 149–153. doi:10.1038/nbt.1775.
TAL Effectors and CRISPR-Cas9 are efficient systems for precise genome modifications in cell lines or model organisms. The table below shows some gene editing applications.
Genome modifications
Description
Genome editing tools
Donor DNA
Gene tagging
Add a fusion tag (e.g. luciferase, GFP) to track an endogenous promoter activity or an endogenous protein expression and location
TALEN or CRISPR
Required
Gene mutagenesis
Introduce point mutations to an endogenous gene
TALEN or CRISPR
Required
Gene knockout
Introduce deletions or insertions (e.g. a selection marker) to knockout an endogenous gene
TALEN or CRISPR
Highly recommended for selection of knockout cell lines
A TAL effector nuclease (TALEN) contains a TALE DNA-binding domain fused to the FokI nuclease. Two TALENs must bind on each side of the target site for FokI to dimerize and cut. TALENs induce a site-specific double-strand break (DSB), which is repaired by non-homologous end joining (NHEJ), which is error-prone and frequently causes small insertions or deletions (“indels”) near the DSB site. These indels often cause frameshift mutations that can efficiently knock a gene out. Alternatively, an exogenous double-stranded donor DNA fragment can be introduced into the genome at the DSB by homologous recombination (HR). TALENs have been used to generate stably modified human embryonic stem cells and induced pluripotent stem cell (IPSCs) clones, and to generate knockout organisms such as mice, C. elegans, and zebrafish.
Figure 1. Illustration of TALEN design
Figure 2. TALEN-mediated gene knockout.(left) TALEN-created DSB are repaired by NHEJ. (right) TALEN-created DSB are repaired by the insertion of selection markers (or other genetic element) from a donor plasmid through HR.
Figure 3. In vitro target DNA cleavage by EGFP-TALENs. (A) The TALEN target plasmid (6110 bp) contains a unique EcoRI site and an eGFP TALEN target site. The two sites are 1066 bp apart. (B) 1 µg of the plasmid was incubated with the indicated enzymes for 30 min at 37°C. 0.5 volume of the digestion reaction was analyzed by agarose gel electrophoresis. * The indicated fragment was checked by PCR, data not shown.
Figure 4. TALENs knockdown eGFP expression. (A) eGFP TALENs expression validation: ~80% confluence HEK293T cells were transfected with 0.8 µg plasmid per well in a 6-well plate. The cells were harvested 48 hrs post-transfection. 1/20th of the cell lysate per well was analyzed for western blot using anti-Flag antibody in an SDS-PAGE (8% resolve gel), with the untransfected cell lysate as the negative control. (B) TALENs knockdown eGFP expression: HEK293T cells in a 6-well plate were co-transfected with EX-EGFP-Lv105 and TALEN plasmids or a control plasmid. EGFP expression was checked under a microscope(Nikon Eclipse Ti, exposure time: 600ms) 48 hours post-transfection.
(A))*The surrogated reporter plasmid was constructed by disrupting a CMV-driven Gaussia luciferase (GLuc) with an in-frame stop codon followed by eGFP TALEN target sequences.
**The donor plasmid contains a promoter-less wild type GLuc, which can replace the interrupted GLuc in the surrogate reporter plasmid and restore the GLuc expression through homologous recombination, which is enhanced by TALEN cleavage.
(B)Figure 5. Restore disrupted Gaussia luciferase function via TALEN-mediated homologous recombination.(A) Illustration of experimental design. (B) HEK293T cells in a 6-well plate were co-transfected with the eGFP-TALEN pair (1 µg), the surrogate reporter plasmid (0.5 µg) and the donor plasmid (0.5 µg). 48hours post-transfection, the restored Gluc activity was determined to evaluate the TALEN function. Internal control SEAP activity was used for normalization.
A key application for TALEs is the targeted activation and repression of target genes in cells by fusing transactivation domains to TALE DNA binding domains. The TALE-TF construct is a powerful tool to selectively modulate gene expression in eukaryotic cells with exquisite specificity. The TALE-TF contains a TALE DNA binding domain fused to the VP64 transcription activator.
Figure 1. Illustration of TALE-TF design
TALE-TF control clone
Figure 2. NTF3 TALE-TF regulates endogenous NTF3 transcription. Endogenous NTF3 transcription activation by TALE-TF: HEK 293T cells transfected with the NTF3 TALE-TF (6 well plate, 1 µg plasmid per well) exhibited a 17-fold increase in the amount of NTF3 mRNA compared to cells transfected with an empty vector. Measurements were performed in triplicate.
Figure 3. Synthetic TALE activators act synergistically to express human genes. (a) Cartoon of a TALE. The indicated amino acids in each repeat recognize the base below. NLS, nuclear localization sequence; VP64/p65, activation domains (ADs). (b) Single TALEs induce target human genes with variable efficiencies. (c) Combinations of TALEs targeting either DNA strand allow for much higher gene induction rates. (Nature Methods. 2013 Vol. 10. No. 3: 207-208)
References
Richter, A. et al. Designer TALEs team up for highly efficient gene induction. Nature Methods. 2013 Vol. 10. No. 3: 207-208
Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nature Methods. 2013 Vol. 10. No. 3: 239-242
Maeder, M.L. et al. Robust synergistic regulation of human gene expression using TALE activators. Nature Methods. 2013 Vol. 10. No. 3: 243-245
GeneCopoeia offers complete custom services for TALE-based targeted genomic modification, including TALEN or TALE-TF design and construction, donor design and construction, functional validations, stable cell line establishment, and transgenic mouse generation.
Advantages
Fast delivery of TAL effector plasmids or mRNA transcripts
Fully sequence-verified and transfection-ready
Functional validations available
Knockin donor plasmid also available
Stable cell line or transgenic mouse services available
Service details
Services
Description
Application
Validation Services
T7 endonuclease I cleavage assay
Chromosomal-level functional validation. It can detect the presence of the indels created by TALEN-mediated NHEJ repairs at the specific target site of the chromosome.
TALEN
qPCR assay
Chromosomal-level functional validation. It can measure the change in expression level of the target gene induced by site-specific TALE-TF transcription activator.
TALE-TF
Donor clone services
Donor clone design and construction
Customized plasmids designed to specifically transfer your gene of interest, selection marker or other genetic element into targeted site through homologous recombination (HR) induced by our genome editing tools.
We offer various donor vector choices with different selection markers and genetic elements built in for your experiment purpose.
TALEN
Stable cell line services
Single colony
Single clone of stable cell line with TALEN-mediated genome modifications.
TALEN
Cell bank
Creating cell bank of monoclonal stable cell line with TALEN-mediated genome modifications.
TALEN
Transgenic mouse services
Transgenic mouse development
Transgenic mice with TALEN-mediated genome modifications
GeneCopoeia offers multiple lentiviral tools for over-expression or knockdown of your gene or microRNA of interest. With the option to buy the tools for producing lentivirus or buy pre-made lentivirus, GeneCopoeia collection of lentiviral tools seek to address your lentiviral research needs.
Available for any construct including ORF cDNA, shRNA, precursor miRNA and miRNA inhibitors
Choice of 100s of expression vectors and fusion tags
Figure 2. H1299 cells (ATCC) in 24-well plates were transduced with the indicated amounts of Standard mCherry lentiviral particles, catalog number LP-MCHR-LV105 in the presence of 5 µg/ml of polybrene. The expression of eGFP was checked with a fluorescence microscope 72d hours post-transduction.
The study of protein subcellular localization is necessary to elucidate protein function. To facilitate protein localization studies inside cells, organelles and tissues, GeneCopoeia offers multiple sets of expression-ready ORF cDNA constructs. These constructs consist of full-length mouse and human cDNA ORFs fused with various tags including eGFP, mCherry, SNAP-Tag™ and HaloTag®.
Subcellular protein localization in live and fixed cells
Monitoring of multiple proteins trafficking in one assay
Protein detection, tracking and characterization
Organelle morphology without antibodies
Co-localization (see figure 1)
Co-localization of two proteins in the same cell using OmicsLink™ ORF expression clones
1
2
3
4
5
Figure 1. TOMM20 (translocase of outer mitochondrial membrane 20 homolog) is located in the mitochondria. LEF1 (Lymphoid enhancer-binding factor 1) is located in the nucleus. Using standard cell and transfection techniques, cells were co-transfected with two plasmids; EX-G0283-M26 (encoding the TOMM20 gene fused with SNAP-Tag™ ) and EX-W0594-M50 (encoding the LEF1 gene fused with HaloTag®). Twenty-four hours after transfection, the cells were labeled with HaloTag® TMR ligand and SNAP Cell BG505 ligand according to the manufacturer’s instructions. The nuclei were stained with DAPI and the subcellular structures of the cells were imaged using fluorescent microscopy: (1) cells labeled with Halo-TMA ligand (red); (2) cells labeled with SNAP Cell BG505 ligand (green); (3) overlay of 1 and 2; (4) cells stained with DAPI; and (5) overlay of 1, 2 and 4.
Validated organelle markers and predicted subcellular localization genes
Click on each organelle name for gene list or Browse all validated organelle markers and predicted subcellular localization genes.
Human full-length ORF expression clones for studying protein localization
