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.
Designer TALEN Technology Information
Precise and extraordinarily flexible
TALEN or TAL effectors are a widely used technology for precise and efficient gene editing in live cells. This genome editing technology is known to function in a variety of host systems, including bacteria, yeast, plants, insects, zebrafish, and mammals. Thermo Fisher Scientific has secured rights to certain proprietary methods related to TALENs, clarifying a path for you to move confidently forward and innovate. We've put together a collected of resources that we hope will give you the confidence to get started and to continuously improve research using designer TALENs.
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 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 TALEN effector proteins are already helping advance a broad range of life science applications, including cell, molecular, and synthetic biology; drug discovery; and biofuels research.
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.
Available effector domains
Nuclease function—Fok1 nuclease pair
Recommended for gene targeting, including:
- Incorporation of exogenous DNA
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 (see figure). 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 GeneArtPrecision 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.
Activator function—activator VP16 or VP64
Recommended for activation of transcription, including:
- Increasing the expression level of endogenous gene isoforms
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 (see figure). 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.
Targeted gene activation can be accomplished with a GeneArt Precision TAL protein fused to a VP16 transcription activator domain.
Recommended for epigenetic repression of transcription, including:
- Heritable knockdown of gene expression
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 (see figure).
Targeted gene repression can be accomplished with a GeneArt Precision TAL protein fused to a Krüppel associated box (KRAB) transcription repressor domain.
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
Recommended for custom-designed steric repression, including:
- Transient knockdown of gene expression
- Target any locus in the genome with the effector domain of your choice
You can specifically target a custom effector to any locus in the genome with a GeneArt Precision TALEN protein fused to the effector domain.
Intended as an introduction to genome editing, the resource guide covers the technologies available today for gene editing, along with methods for design, delivery and detection of edited cells.
Learn more about GeneArt Precision TALs technology in this presentation by Dr. Jon Chesnut, Research Fellow and R&D lead at Thermo Fisher Scientific.
Application Note: Using Sanger sequencing to facilitate CRISPR- and TALEN-mediated genome editing workflows
Learn Sanger sequencing by capillary electrophoresis and Minor Variant Finder Software can be used in a genome editing workflow. We show the results of CRISPR-mediated editoing, but the principles applied here can also be used for ZFN- or TALEN-mediated editing workflows. The simplicity and cost-effectiveness of the workflow and uncomplicated data analysis make Sanger sequencing by capillary electrophoresis a valuable part of any genome editing workflow.
Application Note: Improve genome editing outcomes in biologically relevant cell models
With increasing expansion into research areas of more biological relevance, existing molecular and cellular techniques need to be improved. Invitrogen™ Lipofectamine™ 3000 Transfection Reagent, a new reagent developed to improve delivery and enable use of new technologies, can be used in more relevant systems enabling faster and more reliable outcomes.
Top 10 Tips & Tricks: TAL effector-mediated genome editing
TALEN-based genome editing offers numerous possibilities and our R&D scientist would like to share the tips and tricks they’ve learned along the way.
For Research Use Only. Not for use in diagnostic procedures.