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Now that Twitter enable downloading of the entire tweet archive, Sophien Kamoun retrieved these for me... Another one is here: https://twitter.com/KamounLab/statuses/2764683167 
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Hisano et al., 2013
Artificially designed nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can induce a targeted DNA double-strand break at the specific target genomic locus, leading to the frameshift-mediated gene disruption. However, the assays for their activity on the endogenous genomic loci remain limited. Herein, we describe a versatile modified lacZ assay to detect frameshifts in the nuclease target site. Short fragments of the genome DNA at the target or putative off-target loci were amplified from the genomic DNA of TALEN-treated or control embryos, and were inserted into the lacZα sequence for the conventional blue–white selection. The frequency of the frameshifts in the fragment can be estimated from the numbers of blue and white colonies. Insertions and/or deletions were easily determined by sequencing the plasmid DNAs recovered from the positive colonies. Our technique should offer broad application to the artificial nucleases for genome editing in various types of model organisms.
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Gabsalilow 2013 http://nar.oxfordjournals.org/content/early/2013/02/12/nar.gkt080.long
Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.
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(via T. Lahaye)
Politz et al, 2012 Transcriptional repression is a common approach to control gene expression in synthetic biology applications. Here, an engineered DNA binding protein based upon a transcription activator-like effector (TALE) scaffold was shown to outperform LacI in blocking transcription from a promoter and to repress expression of a downstream gene in an operon.
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(via Tom Schreiber, thanks!)
Meckler et al, 2013 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.
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via Tom Schreiber (thanks!) Perez-Pinera et al, 2013
Mammalian genes are regulated by the cooperative and synergistic actions of many transcription factors. In this study we recapitulate this complex regulation in human cells by targeting endogenous gene promoters, including regions of closed chromatin upstream of silenced genes, with combinations of engineered transcription activator–like effectors (TALEs). These combinations of TALE transcription factors induced substantial gene activation and allowed tuning of gene expression levels that will broadly enable synthetic biology, gene therapy and biotechnology.
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We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target.
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Kim et al, 2013 Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.
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Bochtler 2012
Phytopathogen transcription activator-like effectors (TALEs) bind DNA in a sequence specific manner in order to manipulate host transcription. TALE specificity correlates with repeat variable diresidues in otherwise highly stereotypical 34–35mer repeats. Recently, the crystal structures of two TALE DNA-binding domains have illustrated the molecular basis of the TALE cipher. The structures show that the TALE repeats form a right-handed superhelix that is wound around largely undistorted B-DNA to match its helical parameters. Surprisingly, repeat variable residue 1 is not in contact with the bases. Instead, it is involved in hydrogen bonding interactions that stabilize the overall structure of the protein. Repeat variable residue 2 contacts the top strand base and forms sequence-specific hydrogen bonds and/or van der Waals contacts. Very unexpectedly, bottom strand bases are exposed to solvent and do not make any direct contacts with the protein. This review contains a summary of TALE biology and applications and a detailed description of the recent breakthroughs that have provided insights into the molecular basis of the TALE code.
dromius's insight:
I had overseen that...
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8. A new tool helps researchers manipulate our genes. These TALENs can be customized to seek out and edit any DNA sequence, so it's very specific. It can be used to chop out a bad gene and replace it for a treatment, or can be used in research to understand what specific DNA stretches do in healthy and diseased individuals.
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dromius's insight:
Gao et al (2012)
Several cryptic repeats are formed upstream of the central repeat region providing the bulk of binding energy required for high affinity target binding.
Xanthomonas TALEs (transcription activator-like effectors) are modular proteins characterized by an N-terminal T3S signal (T3SS), a central tandem repeat domain, C-terminal nuclear localization signals (NLSs) and an acidic transcriptional activation domain (AD). The central tandem repeats are nearly identical and are typically composed of 34 highly conserved amino acids, containing repeat-variable diresidues (RVDs) at the 12th and 13th positions that mediate their DNA binding specificity.
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GeneCopoeia offers custom services for designing, creating and validating TALENs, TALE-TFs and other TAL effectors-based targeted genomic modification tools.
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TAL (transcription activator-like) effectors from Xanthomonas bacteria activate the cognate host genes, leading to disease susceptibility or resistance dependent on the genetic context of host target genes. The modular nature and DNA recognition code of TAL effectors enable custom-engineering of designer TAL effectors (dTALE) for gene activation. However, the feasibility of dTALEs as transcription activators for gene functional analysis has not been demonstrated. Here we report the use of dTALEs, as expressed and delivered by the pathogenic Xanthomonas oryzae pv. oryzae (Xoo), in revealing the new function of two previously identified disease-related genes and the potential of one developmental gene for disease susceptibility in rice/Xoo interactions. The dTALE gene dTALE-xa27, designed to target the susceptible allele of the resistance gene Xa27, elicited a resistant reaction in the otherwise susceptible rice cultivar IR24. Four dTALE genes were made to induce the four annotated Xa27 homologous genes in rice cultivar Nipponbare, but none of the four induced Xa27-like genes conferred resistance to the dTALE-containing Xoo strains. A dTALE gene was also generated to activate the recessive resistance gene xa13, an allele of the disease susceptibility gene Os8N3 (also named Xa13 or OsSWEET11, a member of sucrose efflux transporter SWEET gene family). The induction of xa13 by the dTALE rendered the resistant rice IRBB13 (xa13/xa13) susceptible to Xoo. Finally, OsSWEET12, an as yet uncharacterized SWEET gene with no corresponding naturally occurring TAL effector identified, conferred susceptibility to the Xoo strains expressing the corresponding dTALE genes. Our results demonstrate that dTALEs can be delivered through the bacterial secretion system to activate genes of interest for functional analysis in plant. Via Ricardo Oliva
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Transcription activator–like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.
Eli's comment,
February 18, 2013 1:18 PM
if you look at the supplemental table 5 though, you can see that they only looked down to 0.5% limit of detection, at only 13 total off-target sites in bulk cell populations (spread across 11 different TALENs...so only 1 off-target site for most TALENs), and only down to 85% sequence homology. Previous reports have found off-target TALEN activity with as low as 73% homology. While it's encouraging that there are not major widespread off-target effects, this was a very rudimentary search that they performed
Eddie Vedder's comment,
February 20, 2013 12:13 PM
Hi Eli, could you give the references with the off target effects with just 73% of homology? Iddn't see them and would be interested...
Eli's comment,
February 20, 2013 12:19 PM
doi:10.1038/nbt.1927 has two sites (one with 72% homology and one with 74%) and doi:10.1038/nbt.1940 has one site (78% homology)
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(via Tom Schreiber, thanks) Maeder et al, 2013
Artificial activators designed using transcription activator–like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.
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(via Tom Schreiber. Thanks!) Mashimo et al, 2013
TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.
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via Tom Schreiber, thanks.
Sakuma et al, 2013 Transcription activator–like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the ‘Golden Gate TALEN and TAL Effector Kit’ (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.
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http://www.talendesign.org/mojohand_main.php
We present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method.
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Background: Human mitochondrial complex I is composed of 44 subunits with an elaborate assembly mechanism. Results: TALENs were used to knock out complex I subunit NDUFA9 in HEK293T cells, revealing a novel membrane arm subcomplex. Conclusion: NDUFA9 is critical for stabilizing the junction between different arms of complex I. Significance: TALEN-mediated knock-out represents an accessible approach to address protein function in human cells.
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Chen et al, 2013 Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identified germ line mutations using ZFNs whose somatic mutations rates are well below the commonly used threshold of 1%. Guidelines that have previously been proposed to predict optimal ZFN and TALEN target sites did not predict mutagenicity in vivo. However, we observed a significant negative correlation between TALEN mutagenicity and the number of CpG repeats in TALEN target sites, suggesting that target site methylation may explain the poor mutagenicity of some TALENs in vivo. The higher mutation rates and ability to target essentially any sequence make TALENs the superior technology for targeted mutagenesis in zebrafish, and likely other animal models.
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Four rice genes and eight Brachypodium genes were targeted to generate knockout mutations. A protoplast transient assay was developed in both rice and Brachypodium. TALEN-encoding constructs were introduced into protoplasts using polyethylene glycol (PEG). Seven out of eleven TALENs showed mutagenesis frequencies ranging from
Two rice TALENs separated by 1322 bp in the OsBADH2 These results support the idea that TALEN-induced genomic deletions were mediated via NHEJ. Targeted deletions would make possible the selective removal of gene clusters and enables scientists to delete intergenic regions, introns, regulatory elements and noncoding RNAs.
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dromius's insight:
Ansai et al (2013) Abstract: Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrated efficient targeted mutagenesis in medaka (Oryzias latipes), which serves an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homologue of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations, because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1Δ7/Δ7 fish that carried a TALEN-induced frame-shift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N-terminus and 63 amino acids at the C-terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.
[Image: Wikipedia/NARUSE, Kiyoshi]
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dromius's insight:
Nga-Sze Mak et al (2012) abstract: TAL effectors are proteins secreted by bacterial pathogens into plant cells, where they enter the nucleus and activate expression of individual genes. TAL effectors display a modular architecture that includes a central DNA-binding region comprising a tandem array of nearly identical repeats that are almost all 34 residues long. Residue number 13 in each TAL
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