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Chromosomal Contact Permits Transcription between Coregulated Genes - Cell

Chromosomal Contact Permits Transcription between Coregulated Genes - Cell | talens | Scoop.it

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Fanucchi et al, 2013

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins.

To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex.

Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact.

This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.


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Production of Sry knockout mouse using TALEN via oocyte injection - Scientific Reports

Production of Sry knockout mouse using TALEN via oocyte injection - Scientific Reports | talens | Scoop.it

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Kato et al, 2013

Recently developed transcription activator-like effector nuclease (TALEN) technology has enabled the creation of knockout mice, even for genes on the Y chromosome.

In this study, we generated a knockout mouse for Sry, a sex-determining gene on the Y chromosome, using microinjection of TALEN RNA into pronuclear stage oocytes. As expected, the knockout mouse had female external and internal genitalia, a female level of blood testosterone and a female sexually dimorphic nucleus in the brain. The knockout mouse exhibited an estrous cycle and performed copulatory behavior as females, although it was infertile or had reduced fertility. A histological analysis showed that the ovary of the knockout mouse displayed a reduced number of oocytes and luteinized unruptured follicles, implying that a reduced number of ovulated oocytes is a possible reason for infertility and/or reduced fertility in the KO mouse.


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Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - Scientific Reports

Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - Scientific Reports | talens | Scoop.it

Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.


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Conditional targeted genome editing using somatically expressed TALENs in C. elegans - Nature Biotech.

Conditional targeted genome editing using somatically expressed TALENs in C. elegans - Nature Biotech. | talens | Scoop.it

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Cheng et al, 2013

We have developed a method for the generation of conditional knockouts in Caenorhabditis elegans by expressing transcription activator–like effector nucleases (TALENs) in somatic cells. Using germline transformation with plasmids encoding TALENs under the control of an inducible or tissue-specific promoter, we observed effective gene modifications and resulting phenotypes in specific developmental stages and tissues. We further used this method to bypass the embryonic requirement of cor-1, which encodes the homolog of human severe combined immunodeficiency (SCID) protein coronin, and we determined its essential role in cell migration in larval Q-cell lineages. Our results show that TALENs expressed in the somatic cells of model organisms provide a versatile tool for functional genomics.


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Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN™ - PLOS One

Targeted Gene Therapy of Xeroderma Pigmentosum Cells Using Meganuclease and TALEN™ - PLOS One | talens | Scoop.it

Xeroderma pigmentosum group C (XP-C) is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG) located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases,

 

Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells.

Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.

 

 


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Construction and Application of Site-Specific Artificial Nucleases for Targeted Gene Editing - Methods Mol. Biol.

Construction and Application of Site-Specific Artificial Nucleases for Targeted Gene Editing - Methods Mol. Biol. | talens | Scoop.it

Artificial nucleases have developed into powerful tools for introducing precise genome modifications in a wide variety of species. In this chapter the authors provide detailed protocols for rapidly constructing zinc finger nucleases (ZFNs) and TALE nucleases (TALENs) and evaluating their activity for the targeted generation of InDels within the zebrafish genome.


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Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors - PLOS One

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors - PLOS One | talens | Scoop.it

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Wan et al, 2013

Molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism.


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Creating a TALE protein with unbiased 5′-T binding - Biochem. Biophys. Res. Comm.

Creating a TALE protein with unbiased 5′-T binding - Biochem. Biophys. Res. Comm. | talens | Scoop.it

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Tsuji et al, 2013

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.


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Specific reduction of mutant mitochondrial genomes load in patient-derived cells by mitoTALENs - mitochondrion


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dromius's curator insight, November 1, 2013 2:14 PM

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Bacman et al, 2013

Mitochondrial diseases are commonly caused by high levels of heteroplasmy mutations in the mitochondrial DNA (mtDNA). In an attempt to reduce the mutant mtDNA load, we have engineered TAL-effector nucleases (TALEN) to localize to mitochondria. We designed two different mitochondrial-targeted TALENs (mitoTALENs) to bind and cleave two different mtDNA pathogenic mutations: the “common deletion of 5 kbp” (associated with ocular myopathies and Kearns–Sayre syndrome) and a G14459A point mutation in the ND6 subunit of Complex I (associated with optic atrophy and dystonia). The mitoTALENs included a mitochondrial localization signal, a tag in the N-terminus of the mature protein, a 3′UTR from a mitochondrial gene, and a fluorescent marker to select for double transfected cells. All these elements were assembled in a single plasmid. Cybrid cells harboring heteroplasmic levels of the mutated mtDNAs were transfected with the mitoTALENs. MitoTALENs were able to effectively localize to mitochondria and express the expected size protein. Cybrid cells were transfected and flow sorted for the fluorescent markers after 48 h. MitoTALENs were able to reduce the levels of the pathogenic mtDNA common deletion (5 kb deletion) and the levels of the point mutation G14,459A in the respective cybrids, in both cases increasing the levels of WT mtDNA. No significant depletion of total mtDNA was found when quantified by RT-PCR normalized to genomic ß actin. Functional assays for Complex I activity in cybrid lysates showed that the cells harboring the point mutation G14458A that originally were defective in Complex I, were able to recover the activity to control levels. These findings raise the possibility that mitoTALENs can be beneficial to a large subgroup of patients with mitochondrial diseases associated with heteroplasmic mtDNA and may be a viable genetic therapy in the future.

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TALEN-based knockout library for human microRNAs - Nature Struc. & Mol. Biol.

TALEN-based knockout library for human microRNAs - Nature Struc. & Mol. Biol. | talens | Scoop.it

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Kim et al, 2013

Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator–like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2–4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.


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Comparing ZFNs and TALENs for Gene Targeting in Drosophila - Genes Genomes Genetics

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Beumer et al, 2013

Zinc-finger nucleases (ZFNs) have proved successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially higher than for ZFNs, and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.


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TALENSeek: A validated program for identifying genomic engineering sites

TALENSeek: A validated program for identifying genomic engineering sites | talens | Scoop.it

http://geschwindlab.neurology.ucla.edu/node/216

 

We developed automated software, entitled TALENSeek, to identify unique TALEN binding sites of a user-specified gene or genomic locus. The program is outlined in Figure 1, where (a) automated annotation of genetic regions are used to identify start and end sites of a gene using the farthest protein coding regions across all isoforms (b) potential TALEN binding sites are identified; (c) TALEN binding sites are evaluated for uniqueness across the genome; (d) if a binding site is non-unique, the binding sites are shifted into the coding region of the gene and the algorithm iterates from step (b). Chromosomal locations and genomic sequences of the TALEN binding site are output. In addition, chromosomal locations of TALEN binding sites can be visualized in genome browsers. Homology arms to generate gene knock-outs, knock-ins or correction are also output by the program and can be used to generate donor constructs to direct homologous dependent repair.
The TALENSeek program has been applied to generate genome-wide TALEN binding sites for each isoform of each gene in the human genome (hg19; 509,262 unique binding sites generated). Eight TALEN pairs were assembled and demonstrated activity in K562 cells; one of these eight has been used in human embryonic stem cells to generate hetero- and homozygously targeted cell lines. TALENSeek can be easily modified to any species with an available genome sequence. An application of the program to mouse (mTALENSeek) is also currently available.
TALENSeek is a user-friendly bioinformatics tool that allows for the design of unique TALEN binding sites in the genome. TALENs are efficient genome editing tools, which create the possibility of direct modeling of disease-associated mutations in developing human stem cells.


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FairyTALE: a high-throughput TAL effector synthesis platform - ACS Synthetic Biology

FairyTALE: a high-throughput TAL effector synthesis platform - ACS Synthetic Biology | talens | Scoop.it

Here, we introduce fairyTALE, a liquid phase high-throughput TALE synthesis platform capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency at a reagent cost of just $5 per TALE. As proof of concept, we synthesized and tested 90 TALEs, each recognizing 27 bp, without restrictions on their sequence composition. 96% of these TALEs were found to be functional, while sequencing confirmation revealed that the non-functional constructs were all correctly assembled.


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Efficient TALEN Construction for Bombyx mori Gene Targeting - PLOS One

Efficient TALEN Construction for Bombyx mori Gene Targeting - PLOS One | talens | Scoop.it

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Takasu et al, 2013

We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments.

 

 


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Targeted Gene Deletion of miRNAs in Mice by TALEN System - PLOS One

Targeted Gene Deletion of miRNAs in Mice by TALEN System - PLOS One | talens | Scoop.it

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Takada et al, 2013

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

 

 


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