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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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Given that the frequencies of mutation and the extent of toxicities we observe are similar to what we have seen with ZFNs, we expect that TALEN-induced mutations should be efficiently passed through the germ line.
all six TALEN monomers we constructed showed high mutagenesis activities when tested in various pairwise combinations. This suggests that the TALEN framework is also highly robust and effective in zebrafish.
TALENs we made in zebrafish did not show toxicity substantially different from that observed with expression of ZFNs
TALENs may offer potential advantages over ZFNs for mutagenesis of genes in zebrafish and other model organisms such as Caenorhabditis elegans12, because they can be easily and quickly assembled in a modular fashion and can potentially target a greater range of DNA sequences. Thus, we expect that the ability to use both ZFNs and TALENs should enable any researcher to rapidly and easily create targeted mutations in any zebrafish gene of interest.
(Picture from: http://www.digs-bb.de)
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Two papers published today (August 7) in Nature journals describe ways to systematically find such off-target action, which could one day help design gene therapies that avoid collateral damage
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$350 for a kit plate of build-your-own TAL effectors and TAL effector nucleases
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A new technology for genome editing may put the zinc finger nuclease franchise out of business, some believe. Not so fast, say the finger people. Laura DeFrancesco reports.
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"Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. ... Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. ... Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB (double strand breaks) in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. ... great potential for site-specific genome modification in plants and eukaryotes in general"
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It is this repeat domain that is responsible for specific DNA sequence recognition. Each repeat is almost identical with the exception of two variable amino acids termed the repeat-variable diresidues.
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"one needs only to modify the" two variable amino acids per repeat, called "RVDs, to generate designer TALEs"
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The most complete review on TAL effectors from Xanthomonas bacteria to date
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"Additionally, TALE-NT provides tools to design single TAL effectors to bind to a DNA sequence, and to identify binding sites in a DNA sequence for a given TAL effector.
The TALE-NT website is maintained by the Bogdanove laboratory at Iowa State University."
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From
jb
26 TAL effector genes in newly sequenced Xanthomonas oryzae pv. oryzicola strain BLS256, none in Xanthomonas campestris raphani 756C
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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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dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.
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An in vitro method for examining cleavage patterns of zinc-finger nucleases (ZFNs) identifies previously unknown off-target cleavage sites.
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Two papers published today, one in Nature Biotechnology and one in Nature Methods, show that although zinc-finger nucleases are highly specific, the methods previously used to predict where they might go wrong miss the mark.
Good to have an alternative strategy...
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"Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence"
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A TAL effector of "Xanthomonas oryzae pv oryzae (Xoo) transcriptionally activates rice (Oryza sativa) susceptibility gene Xa13 to cause bacterial blight disease. Xa13 encodes an indispensable plasma membrane protein"..."We show that the XA13 protein cooperates with two other proteins"..."to promote removal of copper from xylem vessels, where Xoo multiplies and spreads to cause disease"
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The TAL Effector Technology license was jointly signed with inventors Jens Boch, Ulla Bonas, Thomas Lahaye and Sebastian Schornack, and the Two Blades Foundation (2Blades), which exclusively licensed plant research tool ...
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Another publication describing the use of type IIS restriction enzymes in two sequential cut-ligase reactions to build TAL DNA binding domain repeat arrays
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"Recent elucidation of the basis for specificity in DNA binding by TALEs expedites further discovery and opens the door to biotechnological applications. This article reviews the most significant findings in TALE research, with a focus on recent advances, and discusses future prospects including pressing questions yet to be answered"
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TAL effector - Description: TAL effectors (TALEs) are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species.
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