Precise Genome Engineering With GeneArt® Precision TALs
Custom DNA-Binding Proteins for Locus-Specific Modifications
Transcription activator–like (TAL) effector proteins are used by certain plant pathogens to manipulate cellular processes and suppress immunity in their host cells. The extraordinary specificity of the TAL proteins for their target DNA sequences enables their exploitation for precise genome engineering. GeneArt® Precision TALs Products and Services provide researchers with custom DNA-binding proteins (encoded in Gateway® compatible entry clones) for accurate DNA targeting, providing a means of editing specific loci throughout the genome. These new products will help to advance a broad range of life science applications, including cell, molecular, and synthetic biology; drug discovery; and biofuels research.
How Precision TALs Work
TALs are bacterial proteins identified in Xanthomonas bacteria, a genus of plant pathogens responsible for the natural spread of disease via their type III secretion system . In nature, TAL proteins comprise an average of 15–20 repeats of a 34 amino acid sequence flanked by N- and C-terminal domains. Each repeat unit recognizes a single base of DNA in the plant chromosome; nucleotide specificity is conferred by amino acids 12 and 13 within each repeat. Combinations of these repeats allow a TAL to target a specific locus for activation, repression, or the introduction of a double-stranded break.
An understanding of the composition and function of these repeats has allowed scientists to engineer the way that the TAL protein binds and behaves . What was once used only in nature to facilitate plant infection can now be employed for precise genome engineering and the targeted modulation of gene activity in a diverse group of hosts that extends well beyond plants to include mammalian cells, Drosophila, zebrafish, yeast, algae, and bacteria.
Create Custom Genome-Engineering Tools
GeneArt® Precision TALs provide custom DNA-binding proteins fused to effector domains for accurate DNA targeting and precise genome editing. Similar technologies such as zinc-finger nucleases are limited in their choice of targets. In contrast, the predictability with which Precision TALs bind to an exact DNA sequence makes it possible to target any locus in the genome with a known sequence. The choice of effector domain then determines whether the Precision TAL edits, activates, or represses the targeted gene.
GeneArt® Precision TALs are supplied as Gateway® compatible entry clones (lyophilized DNA) encoding a DNA-binding protein—specific for a customer-submitted DNA sequence—fused to one of several available or customer-designated effector domains (Table 1). The ordering process is simple: you specify the target TAL DNA-binding sequence (19 or 25 nucleotides, starting with a T), as well as the TAL effector domain and the Gateway® compatible vector that best suits your needs. We send you a clone with a verified, optimized sequence approximately 2 weeks after confirming your order.
Table 1. Selected GeneArt® Precision TALs.
|Selected products||TAL*||Effector domain|
|Native TAL Fok1
Truncated TAL Fok1
|Native TAL VP16 activator||Native||VP16 activator|
|Native TAL VP64 activator||Native||VP64 activator|
|Native TAL KRAB repressor||Native||KRAB repressor|
|Native TAL MCS
Truncated TAL MCS
|Multiple cloning site (MCS)
Multiple cloning site (MCS)
|*A native TAL includes the complete backbone, from N to C terminus; a truncated TAL is missing the endogenous activation domain.
Order custom GeneArt® Precision TALs Products and Services .
Introduce Double-Strand Breaks With the Fok1 Domain
Double-strand DNA breaks can be created at a customer-specified locus in the genome using a pair of GeneArt® Precision TALs that have been fused to the Fok1 nuclease (Figure 1A). The breaks induced by the Fok1 nuclease domain are subsequently repaired through two cellular mechanisms: nonhomologous end joining or homologous recombination. The repairs enable gene silencing or targeting at any locus in the genome. Much like the cut-and-paste feature on a computer, Precision TALs can cleave a DNA sequence to induce gene silencing or insert an exogenous DNA fragment into an exact location in the genome.
Figure 1. Three varieties of GeneArt® Precision TALs. GeneArt® Precision TALs are engineered with (A) nuclease function, (B) activator function, or (C) a multiple cloning site (MCS). (A) Double-stranded breaks can be created at a specific gene locus using a pair of Precision TALs fused to the Fok1 nuclease. These breaks are then repaired through two cellular mechanisms—homologous recombination or nonhomologous end joining—producing an edited DNA sequence. (B) Gene activation is accomplished by specifically targeting a transcription activator such as herpes simplex VP16 to the gene of interest. (C) For the effector domain of your choice, a multiple cloning site (MCS) vector allows you to insert any protein-coding sequence of interest. The resulting TAL will deliver that effector in a sequence-specific manner to any site in the genome for which you have a DNA sequence.
Activate Genes With a Transcription Activator Domain
A GeneArt® Precision TAL can also be designed to function as a transcriptional activator that will turn on the gene to which the TAL binds (Figure 1B). These Precision TALs are fused to the herpes simplex VP16 activation domain or to VP64, a tetrameric repeat of VP16’s minimal activation domain. When targeted appropriately within the gene, these Precision TAL activators offer the advantage of expressing all the endogenous splice variants in the proper ratios.
In addition, GeneArt® Precision TALs can be designed to act as repressors that will down-regulate the targeted gene, similar to the function of small interfering RNA (siRNA). For example, a Precision TAL fused to the KRAB repressor domain can function as a locus-specific repressor. Both gene activation and repression can serve to elucidate the role of the gene of interest in specific signaling pathways or other phenotypes.
Customize Your Precision TAL’s Effector Domain
If you would like to deploy an effector domain not currently available (Table 1), we offer a multiple cloning site (MCS) vector that allows you to insert any protein-coding sequence of interest in frame with the sequence for the TAL DNA-binding domain. The resulting Precision TAL fusion protein will deliver the chosen effector to the selected locus in the genome (Figure 1C). We also offer gene synthesis services to generate additional effector domain sequences.
Every Gene Locus Is Within Reach
GeneArt® Precision TALs are custom DNA-binding proteins that are designed for accurate DNA targeting and precise genome editing, with reduced risk of off-target effects. We also offer an array of other gene synthesis products, including GeneOptimizer® technology and GeneArt® cell lines and proteins, as well as a wide variety of cloning vectors.
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.