Transcription Activator-like Effector Proteins Research Articles

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

TALE proteins bind to both active and inactive chromatin

TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

TALE-PvuII fusion proteins--novel tools for gene targeting.

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Creating a TALE protein with unbiased 5′-T binding

Transcription activator-like effectors (TALEs) are convenient tools for genome engineering at specific genomic sites. However, their use is constrained because most TALE binding sites are preceded by a highly conserved 5′ terminal T nucleotide (5′-T). To remove the 5′-T constraint, we substituted tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain for other amino acids. Furthermore, we randomized four amino acid residues of the hairpin loop region of repeat-1. Although point mutation was insufficient to remove the 5′-T constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5′-bases. Our result indicates that the repeat-1 loop region of dHax3 is important for 5′-base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the 5′-T constraint and thus allow targeting of any DNA sequences.

Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?

Programmable DNA binding proteins (PDPs) that selectively bind user-defined nucleotide sequences can navigate protein domains to any desired target within the genome. Different functional domains can be fused to PDPs to achieve (1) activation or repression of transcription, (2) nicking or cleavage of DNA, or (3) modification or removal of epigenetic marks at the targeted genome spot. Given their broad applicability, the development of highly specific, easy-to-use PDPs has been a holy grail of biotechnology since the determination of the 3-D structure of zinc fingers (ZFs) bound to DNA in 1991 (Pavletich and Pabo, 1991).

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

Designer TALEs team up for highly efficient gene induction

Transcription activator–like effectors (TALEs) can be programmed to specifically bind any DNA, which makes these proteins broadly usable for biotechnology applications in many organisms. One can generate highly specific activators, repressors and nucleases by fusing the TALE DNA-binding domain to different functional protein domains1. In this issue, two groups report that TALE activators can achieve even higher levels of gene induction than envisaged before if several are used simultaneously2,3. These results increase the power of TALEs as tools for targeted gene control. TALE proteins have a unique DNA-binding mode. Tandem 34-amino-acid repeats each recognize one DNA base pair in a contiguous sequence, and repeat-variable diresidues (RVDs) at positions 12 and 13 specify which base is bound (Fig. 1a). The last repeat is conserved only for its first part and termed a halfrepeat. This one-to-one relation of RVD and DNA base makes it possible to use a simple pickand-match approach to build artificial TALEs with just about any desired DNA-binding specificity4. Natural TALEs from plant-pathogenic bacteria function as transcriptional activators in plants, but they can be exploited as designer transcription factors in other organisms when fused to broadly active transcription activation domains such as herpes simplex virus VP16 or its tetrameric repeat VP64 (ref. 5). However, the induction rates have often been relatively low so far. The two papers in this issue explore novel ways to achieve efficient synthetic transcriptional activators2,3. Maeder et al.2 generated a total of 70 designer TALEs to induce three human genes: VEGFA, NTF3 and the microRNA cluster MIR302–367. They achieved induction rates of up to 114-fold over basal transcription. One reason might be that they chose DNase I hypersensitive regions within promoters that have an open chromatin conformation and are often targets of endo genous transcription factors. This approach makes sense considering that TALEs could not induce expression of silent Oct4 in murine neural stem cells unless epigenetic modifiers were inhibited by chemicals6. Maeder et al. successfully designed TALEs that bind either DNA strand upor downstream of the natural transcription start site2. This emphasizes that TALEs can support transcription from very different positions. The authors further analyzed whether the total number of repeats per TALE influences its activity. Among TALEs with 14.5–24.5 repeats, those with 16.5– 18.5 repeats had a higher activity in some but not all instances2. In contrast, 10.5 repeats has already been shown to be sufficient for full activity in plants7. In addition, it is not only the repeat number but also the RVD composition8,9 that controls activity: that is, NN (Asn-Asn) and HD (His-Asp) repeats contribute most to overall TALE function. Researchers are well advised to use TALEs with about 17.5 repeats, an obvious choice anyway if specificity needs to be balanced with TALE size. It is known that transcriptional activators can act synergistically to induce much higher expression than what the individual contributions would suggest10,11. So far, these studies have relied mostly on heterologous GAL4– (ref. 10) or zinc finger–activation domain fusions11. To test whether this effect also applies to TALEs, Maeder et al. simply transfected combinations of five or six TALEs for each of the three genes studied (Fig. 1). Indeed, the TALE combinations elevated transcription of the microRNA cluster substantially (from approximately 3to 46-fold individually up to 283-fold together)2, but the effect on the other genes was minor. In the other article, Perez-Pinera et al.3 targeted four human genes (IL1RN, KLK3, CEACAM5 and ERBB2) with 6–8 TALEs each. Annekatrin Richter and Jens Boch are in the Department of Genetics, Martin Luther University Halle-Wittenberg, Halle, Germany. e-mail: jens.boch@genetik.uni-halle.de Figure 1 | 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. a

Quantitative analysis of TALE–DNA interactions suggests polarity effects

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ∼ NN ≫ NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches ‘standard’ RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5′-T. Another surprising observation was that base mismatches at the 5′ end of the target site had more disruptive effects on affinity than those at the 3′ end, particularly in designed TALEs. These results provide evidence that TALE–DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins

TALEs targeting a promoter sequence and fused with a transcription activation domain (TAD) may be used to specifically induce the expression of a gene as a potential treatment for haploinsufficiency. This potential therapeutic approach was applied to increase the expression of frataxin in fibroblasts of Friedreich ataxia (FRDA) patients. FRDA fibroblast cells were nucleofected with a pCR3.1 expression vector coding for TALEFrat#8 fused with VP64. A twofold increase of the frataxin mRNA (detected by quantitative reverse transcription-PCR (qRT-PCR)) associated with a similar increase of the mature form of the frataxin protein was observed. The frataxin mRNA and protein were also increased by this TALE in the fibroblasts of the YG8R mouse model. The addition of 5-aza-2′-deoxycytidine (5-Aza-dC) or of valproic acid (VPA) to the TALE treatment did not produce significant improvement. Other TADs (i.e., p65, TFAP2α, SRF, SP1, and MyoD) fused with the TALEFrat#8 gene did not produce a significant increase in the frataxin protein. Thus the TALEFrat#8-VP64 recombinant protein targeting the frataxin promoter could eventually be used to increase the frataxin expression and alleviate the FRDA symptoms.

Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Variable Di-Residues

The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths – from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.

Transcription Activator-Like Effector Proteins Induce the Expression of the Frataxin Gene

Genes encoding transcription activator-like effector (TALE) proteins may be engineered to target specific DNA sequences. TALEs fused with a transcription activator can be used to specifically induce the expression of a gene. This could lead to completely new therapies for several diseases. We have applied this potential therapeutic approach to Friedreich ataxia (FRDA), as an example. FRDA is due to reduced expression of frataxin because of elongation of a trinucleotide (GAA) repeat in intron 1. Our aim was to develop a potential treatment for FRDA by increasing the expression of the frataxin gene. We engineered 12 TALE genes (TALE(Frat)) encoding TALE(Frat) proteins, each specifically targeting different 14-bp DNA sequences within the proximal region of the human frataxin promoter. When the genes encoding these TALE(Frat) proteins were fused with a transcription activator, that is, four VP16 peptides (i.e., VP64), the resulting TALE(Frat)-VP64 proteins induced the expression of an mCherry reporter gene fused to a mini-cytomegalovirus promoter able to be activated by the insertion of the frataxin proximal promoter upstream to the minipromoter. These TALE(Frat)-VP64 proteins also increased, by 2- to 3-fold, frataxin gene expression (detected by qRT-PCR) in the cells. We conclude that TALE(Frat) proteins targeting the frataxin promoter may be used to increase the expression of frataxin mRNA and potentially could alleviate the symptoms of Friedreich ataxia. TALE methodology opens a new field of research, which could be used to develop TALE proteins to treat other diseases by inducing the expression of specific genes.

Modular recognition of nucleic acids by PUF, TALE and PPR proteins

Sequence specific binding of DNA and RNA is of fundamental importance in the regulation of cellular gene expression. Because of their modular structure repeat domain proteins are particularly well suited for these processes and have been widely adopted throughout evolution. Detailed biochemical and structural data has revealed the key residues responsible for recognition of RNA by Pumilio and FBF homology (PUF) repeat proteins and shown that the base specificity can be predicted and re-engineered. Recent work on the DNA-binding properties of transcription activator-like effector (TALE) proteins has shown that their specificity also relies on only a few key residues with a predictable code that can be used to design new DNA-binding proteins. Although less well understood, pentatricopeptide repeat (PPR) proteins contain motifs that appear to contribute to RNA recognition and comparisons to TALE and PUF proteins may help elucidate the code by which they recognize their RNA targets. Understanding how repeat proteins bind nucleic acids enables their biological roles to be uncovered and the design of engineered proteins with predictable RNA and DNA targets for use in biotechnology.

Targeting DNA With Fingers and TALENs.

Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1,2 Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3,4,5 ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6 However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7,8,9,10 Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy.

Computational Modeling of the Binding Mode of Transcription Activator-Like Effectors to DNA

Transcription activator-like effectors (TALEs) form a novel class of DNA-binding proteins with predictable specificity. The TALE protein for avrBs3 is composed of 17.5 repeat domains of 34 amino acid residues, and each repeat domain recognizes one specific base-pair. The specificity is encoded by the repeat-variable diresidue (RVD) of each repeat domain, and this modular nature of DNA base recognition enables useful applications in biotechnology. However, there is very little available structural information about the binding mode and the specificity. In this study, we attempt to generate possible binding modes of the TALE with avrBs3 using protein structure modeling, protein-DNA docking, and molecular dynamics simulation. We discuss the observed binding specificities in terms of the molecular interactions found in the models.

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

Assembly of Designer TAL Effectors by Golden Gate Cloning

Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.