TALEN technology overview
Transcription activator–like (TAL) effector proteins are produced by bacteria in the genus Xanthomonas, which are widely distributed plant pathogens. Natural TAL effectors bind to specific sequences of host DNA, altering the infected plant’s gene expression in ways that further the disease process. The natural TAL effector proteins have two distinct domains: an effector domain and an extraordinarily specific DNA-binding domain. The structure of the DNA-binding domain can be manipulated to produce a protein domain that binds specifically to any DNA sequence in the genome. These specifically modified DNA-binding protein domains can then be linked to a custom effector domain (e.g., a nuclease, or a transcription activator or repressor) to create a chimeric protein capable of precisely targeted DNA manipulation. This sequence-targeting technology is known to function in a variety of host systems, including bacteria, yeast, plants, insects, zebrafish, and mammals. Whether through targeted nucleases for genome engineering, or by precisely directed moderators of gene expression, researcher-designed TAL effector proteins are already helping advance a broad range of life science applications, including cell, molecular, and synthetic biology; drug discovery; and biofuels research.
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Genome editing using engineered DNA-binding domains
GeneArt® TALs are derived from Xanthomonas TAL effectors, the DNA-binding domain of which consists of a variable number of amino acid repeats. Each repeat contains 33–35 amino acids and recognizes a single DNA base pair. The DNA recognition occurs via 2 hypervariable amino acid residues at positions 12 and 13 within each repeat, called repeat-variable di-residues (RVDs). TAL effector repeats can be assembled modularly, varying the RVDs to create a TAL protein that recognizes a specific target DNA sequence. Linking the repeats is straightforward, and long TAL effectors can be designed to specifically target any locus in the genome. Watch the animated video to see how these work.
Nuclease function—Fok1 nuclease pair
(Fok1 nuclease pair)
Recommended for gene targeting, including:
Double-stranded DNA breaks can be created at your specified genomic locus by using a pair of GeneArt® TAL proteins that have been fused to the Fok1 endonuclease (Figure 1). Using a pair of TAL proteins for the targeting reduces off-target effects. The breaks induced by the Fok1 nuclease domain are subsequently repaired through either of two endogenous cellular mechanisms: nonhomologous end joining (NHEJ), or homology-directed repair (HDR). NHEJ is prone to errors and often introduces a frameshift mutation when it occurs within the coding sequence of a protein-coding gene, effectively silencing the gene. Homologous DNA “donor sequences” can be used with HDR to introduce a defined new DNA sequence. Consequently, a GeneArt® Precision TAL or GeneArt® PerfectMatch TAL protein fused to a Fok1 endonuclease can be used to induce gene silencing or to accurately insert an engineered DNA fragment into an exact location in the genome.
|Figure 1. Designing target sites for customized TAL effectors for maximal binding. (A) GeneArt® Precision TALs encode a DNA-binding protein specific to a customer-submitted sequence, fused to a FokI nuclease domain for genome editing. The sequence targeted by our first-generation TAL effectors must have a T at its 5’ end and spacing between forward and reverse TAL effectors must be 13–18 bp for proper pairing of the FokI nucleases and creation of the double-strand break. (B) GeneArt® PerfectMatch TALs eliminate the 5’ T constraint of GeneArt® Precision TALs. GeneArt® PerfectMatch TALs allow targeting of any sequence across the genome; 15–16 bp spacing between the two TAL effectors is optimal for GeneArt® PerfectMatch TALs.|
Activator function—activator VP16 or VP64
Transcription (Activator vp16 or vp64)
Recommended for activation of transcription, including:
A GeneArt® Precision TALEN™ effector can also be designed to function as a transcriptional activator that will increase transcription of a gene near the TAL effector’s DNA-binding site (Figure 2). To create this site-specific gene activator, a Precision TAL DNA-binding domain is fused to a herpes simplex VP16 activation domain or to VP64, a tetrameric repeat of the VP16 activation domain. When targeted appropriately, these GeneArt® Precision TAL activators offer the advantage of expressing all the endogenous splice variants of the target gene in the naturally occurring ratios.
Figure 2. Targeted gene activation can be accomplished with a GeneArt® Precision TAL protein fused to a VP16 transcription activator domain.
repression of transcription
Recommended for epigenetic repression of transcription, including:
GeneArt® Precision TALs can be designed to act as repressors that will down-regulate a targeted gene. To create this site-specific gene repressor, a Precision TAL DNA-binding domain is fused to a Krüppel associated box (KRAB) domain, a potent repressor of transcription.
Both gene activation and repression have been used to reveal the roles played by specific gene products in signaling pathways or in the expression of various other phenotypes.
Custom function—multicloning site (MCS) vector
and custom design
Recommended for custom-designed steric repression, including:
Figure 4. You can specifically target a custom effector to any locus in the genome with a GeneArt® Precision TALEN™ protein fused to the effector domain.
Limitless choice of targets
Your choice of targets is practically unlimited. The predictability with which GeneArt® Precision TAL effectors bind to exact DNA sequences makes it possible to target any sequence in the genome. The choice of the effector domain then determines whether the TAL effector edits, activates, or represses the targeted gene.
GeneArt® TAL effectors are supplied as lyophilized DNA in the form of Gateway® entry clones that contain DNA specifying a DNA-binding protein domain—specific for the DNA sequence you submit—fused to one of several available effector domains or to a custom effector domain.
If you want to incorporate an effector domain that is not currently available from us, we offer an MCS (multiple cloning site) sequence in place of the effector domain sequence, an option that allows you to insert any protein-coding sequence. The resulting TAL protein will deliver that effector in a sequence-specific manner anywhere in the genome. Additionally, we provide gene synthesis services to generate any effector domains for which you don’t have a template.
GeneArt® Precision TALs and GeneArt® PerfectMatch TALs are supplied as Gateway® entry clones with sequences that encode a DNA-binding protein specific for the DNA sequence you specify, fused to the effector domain you specify.
Monitoring TALEN-based genome editing success
Detect locus-specific double-stranded breaks
The GeneArt® Genomic Cleavage Detection Kit offers a simple, reliable, and rapid method for detection of locus-specific double-stranded break formation. The assay measures the efficiency at which genome editing tools such as CRISPRs, TALs, and zinc finger nucleases cleave at a given locus. This cleavage is easily detectable and quantifiable by gel analysis.
GeneArt® PerfectMatch TALs deliver effective genome editing
Cleavage efficiency assay shows GeneArt® PerfectMatch TALs function as well as or better than first-generation TAL effectors. GeneArt® PerfectMatch TALs' function is equal to or better than current TAL effectors in 293FT cells (A) and HeLa cells (B) when targeting sequences of forward and reverse TAL effectors are preceded by different (non-identical) bases. The red arrowheads point to the cleavage products by GCD enzyme if multiple bands were shown in a GCD assay.
Repression of genes
Repression of genes. Repression of endogenous Sox2: mRNA levels (relative to control) by TAL repressor. The MCS vector as a binding control and pcDNA3 as vector-only control.
Optimal knockdown and reduced off-targets
Optimal knockdown and reduced off-targets. Time course of repression on endogenous genes Sox2 in 293FT cells. TAL repressors epigenetically repress genes in a heritable and persistent manner. 293FT cells were transfected with the indicated plasmids. Cells were harvested at different time points, and mRNA levels of the target genes were quantitated by qRT-PCR and normalized to β-actin.
Maximize the efficiency of genetic modifications
Cleavage efficiency improvements of GeneArt® TALs and CRISPRs following transfection using Lipofectamine® 3000 reagent. The TALENs and CRISPRs targeted the AAVS1 locus in (A) U2OS and (B) HepG2 cell lines. Cleavage was assayed using a GeneArt® Genomic Cleavage Detection Kit.
- Download the application note, Lipofectamine® 3000—Improve genome editing outcomes in biologically relevant cell models
Silencer® Select siRNA for a genome-wide screen resulted in new Parkinson’s disease targets
This Nature publication from NCATS, NIH, demonstrates the utility of a Silencer® Select Genome-Wide siRNA library for arrayed high-throughput screening in effectively identifying novel drug targets for Parkinson’s disease. The workflow adopted for this effort also incorporated our other products Lipofectamine® RNAiMAX Transfection Reagent and Gibco® media. Final validation of lead drug targets was accomplished by creating knockout cell lines using the TAL-effector endonuclease (TALEN) technology.
Q. How specific is TALEN™? Off-site targeting?
A. A recent paper by Prashant Mali in Nature Biotechnology shows that TALEN™ are tolerant to 1–2 mismatches, but less tolerant to a large majority of 3 bp mismatches.
Q. Are TALEN™-meditated KO or KI strains considered to be GMO?
A. Yes, KO and KI involves editing the native genetic code by either mutating or deleting an encoded message or inserting a new piece of information at a desired site. Although this does manipulate the native genetic information, this technology—when used in a responsible manner—has very useful applications, including engineering yeasts for insulin production or engineering cells for more economically and clinically valuable products.
Q. How big is the TALEN™ vector construct?
A. 3.3 kb.
Q. Can TALEN™ be delivered by retroviruses?
A. Yes. If viral-based delivery is your preferred option, we recommend adenoviral systems over lentiviral systems for TAL delivery.
Q. Can TALEN™ recognize degenerate binding sites?
A. By careful designing they can be engineered to be very specific. Note: Recent publications show that 1–3 bp mismatches in target DNA sequences can be tolerated to a large extent.
Q. How fast can I get TALEN™ made so that I can do my KO or KI experiments?
A. Manufacturing takes place typically within 2 weeks after your order has been placed.
Q. What is the best delivery method for TALEN™? Transfection, electroporation, or retroviral delivery?
A. mRNA and DNA are best delivered via lipid-based transfection for standard test cell lines (i.e., 293, HeLa, etc.). mRNA delivery also reduces the risk of transgene integration. For stem cells, electroporation is the best option.
For Research Use Only. Not for use in diagnostic procedures.