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Rescooped by Rachel Younghye Seo from TAL effector science
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Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases (TALENs) - Genetics

Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases (TALENs) - Genetics | BIOTECH | Scoop.it

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dromius's curator insight, January 5, 2013 7:13 AM

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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Surrogate reporters for enrichment of cells with nuclease-induced mutations : Nature Methods

Surrogate reporters for enrichment of cells with nuclease-induced mutations : Nature Methods | BIOTECH | Scoop.it

"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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Rescooped by Rachel Younghye Seo from TAL effector science
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Characterization and DNA-Binding Specificities of Ralstonia TAL-like Effectors - Mol. Plant

Characterization and DNA-Binding Specificities of Ralstonia TAL-like Effectors - Mol. Plant | BIOTECH | Scoop.it

via Tom Schreiber, thanks!

Li et al 2013

Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotides, HN interacts specifically with A and G nucleotides, and NP specifically binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.


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dromius's comment, March 24, 2013 9:01 AM
THIS MANUSCRIPT HAS BEEN RETRACTED BY THE AUTHORS!
Nicolas Denancé's comment, March 24, 2013 10:30 AM
Thanks Dromius for the info. It is always bad news when scientists retract their published work. Is it because they absolutely wanted to be the first to publish data on Ralstonia solanacearum TAL effectors so that they did not check/analyzed their results enough? Sometimes I feel sad because of this publication race. Is it really good for Science?
dromius's comment, March 24, 2013 11:36 AM
There are many reasons/motivation for rushing into publication. It will be interesting to see what follows. The webpage of the journal is not clear about that. It can be an amended version or a retraction note or a new paper...I appreciate that they retracted it rather quickly, as this prevents it from being archived in the heads of people and in reviews.
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A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly - Nuc l. Acids Res.

A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly - Nuc l. Acids Res. | BIOTECH | Scoop.it

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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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