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TAL effector science
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infos on novel DNA-binding proteins of bacteria and their biotech use
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dromius
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The ability to introduce targeted, tailored changes into the genomes of several species will make it feasible to ask more precise biological questions. 
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Analysis of the unassembled short reads failed to find any evidence of TAL effectors in either U.S. strain genome, while the same analysis performed on sequences from non-U.S. X. oryzae strains demonstrated high coverage of TAL sequence ... In addition, two previous reports demonstrated that TAL effector probes do not hybridize to DNA blots from U.S. strains. A BLASTN search revealed that the other predicted type III-secreted effectors reported in X. oryzae genomes ... were nearly all represented in both U.S. X. oryzae sequences, with the exception of those in the XopU and XopO families.
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(Article in Chinese) This article reviews the findings and functions of TAL effectors, the binding specificity and recognition code between TAL-effectors and host target genes. The possible applications and future prospects of the molecular recognition code have been discussed.
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Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, an emerging and destructive disease worldwide. Identification of key virulence factors is prerequisite for understanding the pathogenesis of Xoc. The 13 mutants with attenuated virulence but retained ability to induce an HR (intact type III secretion system) included a tal-C10c-like transcriptional activator-like TAL effector.
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Nature Biotechnology journal featuring biotechnology articles and science research papers of commercial interest in pharmaceutical, medical, and environmental sciences.
"Disruptive technologies must be efficient, robust, universally adoptable and scalable: any gene, any position for whole genomes and for everyone. No limit, just talent."
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"Generating and applying new knowledge from the wealth of available genomic information is hindered, in part, by the difficulty of altering nucleotide sequences and expression of genes in living cells in a targeted fashion. Progress has been made in engineering DNA binding domains to direct proteins to particular sequences for mutagenesis or manipulation of transcription; however, achieving the requisite specificities has been challenging. Transcription activator–like (TAL) effectors of plant pathogenic bacteria contain a modular DNA binding domain that appears to overcome this challenge. Comprising tandem, polymorphic amino acid repeats that individually specify contiguous nucleotides in DNA, this domain is being deployed in DNA targeting for applications ranging from understanding gene function in model organisms to improving traits in crop plants to treating genetic disorders in people." (Image from publication)
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From
jb
"Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. ... Though some X. campestris pv. raphani strains and others Via Kamoun Lab @ TSL
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Targeted genetic modification (TagMo) techniques.., which uses engineered nucleases to create DNA double-stranded breaks at specific genomic locations. This activates DNA repair mechanisms, which genetic engineers can use to alter the target gene. If, for instance, a DNA fragment is provided that has sequence similarity with the site at which the chromosome is broken, the repair mechanism will use this fragment as a template for repair through homologous recombination. In this way, any DNA sequence, for instance a bacterial gene that confers herbicide resistance, can be inserted at the site of the chromosome break. TagMos can also be used without a repair template to make single-nucleotide changes. In this case, the broken chromosomes are rejoined imprecisely, creating small insertions or deletions at the break site that can alter or knock out gene function.
Genetic modification would not necessarily involve transfer of DNA from another species. TagMo technology would, therefore, challenge regulatory policies both in the USA and, even more so, in the European Union (EU).
"Public failures will probably ensue if TagMo crops slip into the market under the radar without adequate oversight"
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Cellectis bioresearch, the genome customization specialist and a commercial subsidiary of Cellectis, and Recombinetics, a specialist in biogenetics, announced today that they have entered a development and license agreement on the creation of transgenic large livestock.
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![Assembly of designer TAL effectors by Golden Gate cloning [PLoS One. 2011] | TAL effector science | Scoop.it](https://img.scoop.it/fnL2mwADhJ-U5gRhw-eOtjl72eJkfbmt4t8yenImKBVvK0kTmF0xjctABnaLJIm9)
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
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The CCR5 protein is a G protein-coupled receptor which functions as a chemokine receptor and predominantly expressed on immune T cells, macrophages, dendritic cells and microglia.
CCR5-Δ32 is a deletion mutation found in about 10% of people of Northern Europe (at least one copy) and in those of Northern European descent. Individuals with the Δ32 allele of CCR5 are healthy, suggesting that CCR5 is largely dispensable.
The CCR5-Δ32 mutation appears to protect against HIV and smallpox (Variola virus).
(source: Wikipedia, modified)
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"This appears to be the first demonstration that TALENs can produce heritable mutations in a vertebrate organism. Based on our results, it is plausible that this technology could be applied to other model organisms, especially those in which gene-targeting methods are still lacking or very preliminary."
"Compared with zinc-finger proteins4, 5, TAL effectors permit more predictable and specific binding to target DNA6, and therefore allow researchers to engineer genomes precisely without the need for laborious screening to identify a DNA binding domain with the requisite specificity."
"The NK RVD has been reported to bind to G more specifically than NN does" "Survival rates were similar for embryos injected with mRNAs encoding either NN- or NK-containing TALENs (Fig. 1b). However, the NN embryos exhibited a noticeably higher mutation rate than the NK embryos"
(Image: http://ec.europa.eu/research/health/genomics/newsletter/issue2/newprojects_en.htm)
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Gene-editing nucleases can make targeted and precise changes to an organism's genome. This has opened up new possibilities for the study of gene function
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Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3.SRDX protein efficiently repressed the transcription of the RD29A::LUC transgene and endogenous RD29A gene in Arabidopsis. Genome wide expression profiling showed that the chimeric repressor also inhibited the expression of several other genes that contain the designer TALE-target sequence in their promoters. Our data suggest that TALEs can be used to generate chimeric repressors to specifically repress the transcription of genes of interest in plants. This sequence-specific transcriptional repression by direct on promoter effector technology is a powerful tool for functional genomics studies and biotechnological applications.
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Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees, and is responsible for emerging diseases in Europe. It comprises seven pathovars (pruni, corylina, juglandis, populi, poinsettiicola, celebensis and fragariae), each exhibiting characteristic disease symptoms and distinct host specificities.
Among X. arboricola pathovars, it should be noted that X. arboricola pv. corylina is the only pathovar that contains homologues of the [TAL effector] avrBs3 gene. (Image: Hazelnut blight, source: plantwise.org)
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"In 2011 alone, TALENs have successfully rendered site-specific mutations, some heritable, in yeast, ESCs, iPSCs, C. elegans, rats, plants, and zebrafish. Compared to ZFNs, efficiency and off-target effects occur at similar, or slightly improved, rates"
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"A simple episomal fluorescent reporter for flow cytometric enrichment of cells with zinc-finger nuclease- or TALE nuclease-induced genomic modifications is described.... Transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry."
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"Zinc-finger nucleases (ZFN) have room for improvement, though; use of other modular DNA-binding proteins called TAL effectors suggests that TAL effector nucleases (TALEN) might be even easier to predict and engineer."
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Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
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Owing to the peculiar properties of their DNA-binding specificity, transcription activator-like effectors (TALEs) from the plant pathogen Xanthomonas can be engineered to bind virtually any DNA sequence. Jaenisch and colleagues exploit this property to drive targeted modifications in both human embryonic stem cells and induced pluripotent stem cells (Nat. Biotechnol. 29, 731–734; 2011), in which genetic engineering by homologous recombination is inefficient.
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We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites.
Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp.
Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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"we demonstrate that three functionally distinct" TAL binding "boxes targeted by separate TAL effectors retain their function and specificity when combined into one promoter. Given that many economically important" plant pathogenic pathogenic xanthomonads" deliver multiple TAL effectors, the engineering of Resistance genes capable of recognizing multiple TAL effectors provides a potential approach for engineering broad spectrum and durable disease resistance"
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"TALENs (TAL Effector Nucleases) to disrupt the rat IgM locus, creating heritable mutations that eliminate IgM function. Our results establish the use of TALEN technology for in vivo gene knockout in mammals"
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"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."
"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"
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