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TAL effector
Curated by Guogen Yang
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Root Systems Biology: Integrative Modeling across Scales, from Gene Regulatory Networks to the Rhizosphere

Root Systems Biology: Integrative Modeling across Scales, from Gene Regulatory Networks to the Rhizosphere | TAL effector | Scoop.it

Genetic and genomic approaches in model organisms have advanced our understanding of root biology over the last decade. Recently, however, systems biology and modeling have emerged as important approaches, as our understanding of root regulatory pathways has become more complex and interpreting pathway outputs has become less intuitive. To relate root genotype to phenotype, we must move beyond the examination of interactions at the genetic network scale and employ multiscale modeling approaches to predict emergent properties at the tissue, organ, organism, and rhizosphere scales. Understanding the underlying biological mechanisms and the complex interplay between systems at these different scales requires an integrative approach. Here, we describe examples of such approaches and discuss the merits of developing models to span multiple scales, from network to population levels, and to address dynamic interactions between plants and their environment.


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Targeted genome modification of crop plants using a CRISPR-Cas system

Although genome editing technologies using zinc finger nucleases (ZFNs)1 and transcription activator-like effector nucleases (TALENs)2 can generate genome modifications, new technologies that are robust, affordable and easy to engineer are needed. Recent advances in the study of the prokaryotic adaptive immune system, involving type II clustered, regularly interspaced, short palindromic repeats (CRISPR), provide an alternative genome editing strategy3. Type II CRISPR systems are widespread in bacteria; they use a single endonuclease, a CRISPR-associated protein Cas9, to provide a defense against invading viral and plasmid DNAs4. Cas9 can form a complex with a synthetic single-guide RNA (sgRNA), consisting of a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA. The sgRNA guides Cas9 to recognize and cleave target DNA. Cas9 has a HNH nuclease domain and a RuvC-like domain; each cleaves one strand of a double-stranded DNA. It can be used as an RNA-guided endonuclease to perform sequence-specific genome editing in bacteria, human cells, zebrafish and mice5, 6, 7, 8, 9, 10, 11. Here we show that customizable sgRNAs can direct Cas9 to induce sequence-specific genome modifications in the two most widely cultivated food crops, rice (Oryza sativa) and common wheat (Triticum aestivum).

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PLOS ONE: A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library

PLOS ONE: A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library | TAL effector | Scoop.it

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Zhang et al, 2013

Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.


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Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs - Cell Regeneration

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Ru et al, 2013

In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.


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Fangjun JiaFang-Jun's curator insight, June 19, 2013 6:56 PM

Not sure whether TAT-TALEN proteins are capable of penetrating cells. Typically, TAT works well to a small peptide while TALEN a larger protein.

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A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta - Plant Mol. Biol.

A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta - Plant Mol. Biol. | TAL effector | Scoop.it

(via T. Lahaye, thanks...cant beat his speed :))

Johnson et al, 2013

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase® reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases’ cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established ‘QQR’ zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.


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Nucl. Acids Res.: Quantitative analysis of TALE–DNA interactions suggests polarity effects (2013)

Nucl. Acids Res.: Quantitative analysis of TALE–DNA interactions suggests polarity effects (2013) | TAL effector | Scoop.it

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.

 

Joshua F. Meckler, Mital S. Bhakta, Moon-Soo Kim, Robert Ovadia, Chris H. Habrian, Artem Zykovich, Abigail Yu, Sarah H. Lockwood, Robert Morbitzer, Janett Elsäesser, Thomas Lahaye, David J. Segal, and Enoch P. Baldwin


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Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding -Trends in Biotechnology

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding -Trends in Biotechnology | TAL effector | Scoop.it

(via T. Lahaye)

Podevin et al, 2013

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding.


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TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants - Journal of Genetics and Genomics

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Chen & Gao 2013

 

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs) consisting of an engineered specific transcription activator-like effector (TALE) DNA binding domain and a FokI cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement.


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Plant Genome Engineering with Sequence-Specific Nucleases - Annual Review of Plant Biology

Voytas 2013

 

Recent advances in genome engineering provide newfound control over a plant’s genetic material. It is now possible for most bench scientists to alter DNA in living plant cells in a variety of ways, including introducing specific nucleotide substitutions in a gene that change a protein’s amino acid sequence, deleting genes or chromosomal segments, and inserting foreign DNA at precise genomic locations. Such targeted DNA sequence modifications are enabled by sequence-specific nucleases that create double-strand breaks in the genomic loci to be altered. The repair of the breaks, through either homologous recombination or nonhomologous end joining, can be controlled to achieve the desired sequence modification. Genome engineering promises to advance basic plant research by linking DNA sequences to biological function. Further, genome engineering will enable plants’ biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population.

Expected final online publication date for the Annual Review of Plant Biology Volume 64 is April 29, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

 


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Designer TALEs team up for highly efficient gene induction - Nature Methods

Richter & Boch, 2013

 

Transcription activator–like effectors (TALEs) can be programmed to specifically bind any DNA, which makes these proteins broadly usable for biotechnology applications in many organisms. One can generate highly specific activators, repressors and nucleases by fusing the TALE DNA-binding domain to different functional protein domains1. In this issue, two groups report that TALE activators can achieve even higher levels of gene induction than envisaged before if several are used simultaneously2, 3. These results increase the power of TALEs as tools for targeted gene control.


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Quantitative assay for TALEN activity at endogenous genomic loci

Quantitative assay for TALEN activity at endogenous genomic loci | TAL effector | Scoop.it

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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Characterization and DNA-Binding Specificities of Ralstonia TAL-like Effectors - Mol. Plant

Characterization and DNA-Binding Specificities of Ralstonia TAL-like Effectors - Mol. Plant | TAL effector | 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 TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models - Cell Stem Cell

A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models - Cell Stem Cell | TAL effector | Scoop.it

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Synthetic nucleases for genome engineering in plants: prospects for a bright future - Plant J.

Synthetic nucleases for genome engineering in plants: prospects for a bright future - Plant J. | TAL effector | Scoop.it

Puchta & Fauser, 2013

By inducing double-strand breaks (DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a way to target DSBs to specific sites. With the development of modified meganucleases and synthetic DNA binding domains, this limitation was overcome. Domains derived from zinc finger transcription factors or transcription activator-like effectors can be designed to recognise almost any DNA sequence. By fusing these domains to the endonuclease domains of a class II restriction enzyme, an active endonuclease dimer can be formed that introduces a site-specific DSB. Recent studies demonstrate that gene knock-outs via nonhomologous end joining or gene modification via homologous recombination are becoming routine in many plant species. By setting a single genomic DSB, the complete knock-out of a gene, the sequence-specific integration of foreign DNA, or the subtle modification of individual amino acids in a specific protein domain can be achieved. The induction of two or more DSBs allows for complex genomic rearrangements such as deletions, inversions, or the exchange of chromosome arms. The potential of controlled genome engineering in plants is tremendous. The recently discovered RNA-based CRISPR/Cas9 system as new tool to induce multiple DSBs or sophisticated technical applications, such as the in planta gene targeting system, are further steps in this development. At the moment, the focus still lies on the engineering of single genes; in the future, the engineering of whole genomes will become an option.


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Clem Stanyon's curator insight, October 2, 2013 6:02 PM

I've just recently started working in the field (no pun intended) of plant genetic engineering and things are not quite so rosy as this makes out; plants are far more likely to repair dsDNA damage by a process called non-homologous end joining (NHEJ) than by homologous recombination (HR); the former process just sticks broken ends together, virtually willy-nilly, while the latter substitutes like-for-like, as determined by DNA sequence homology.

The ratio between NHEJ and HR in yeast, which does HR very effectively by all estimates, is probably about 1:1 - 50% of events are by HR. In plants, the ratio is more like 100:1 - ~1% of events are repaired by HR. The dreaded and annoying off-target effects in plants, therefore, are a far, far higher proportion of the total, so even a highly effective and very specifically targeting dsDNA break promoter like the Cas9/CRISPR system won't be able to prevent NHEJ from swamping the HR events. All is not lost, however, as there are ways to make the proetome of any organism dance to one's own tune, if one has the right instrument...

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Targeted Gene Disruption in the Xenopus tropicalis Genome using Designed TALE Nucleases - Zool. Sci.

Targeted Gene Disruption in the Xenopus tropicalis Genome using Designed TALE Nucleases - Zool. Sci. | TAL effector | Scoop.it

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Nakajima et al, 2013

To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.

 


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Trends in Biotechnology - ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.

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TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins - Trends Cell Biol

TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins - Trends Cell Biol | TAL effector | Scoop.it

 

(via Tom Schreiber, thx)

Doyle et al, 2013

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria of the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA-binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting.


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Compact designer TALENs for efficient genome engineering - Nature Comm.

Compact designer TALENs for efficient genome engineering - Nature Comm. | TAL effector | Scoop.it

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Beurdeley et al, 2013

Transcription activator-like effector nucleases are readily targetable ‘molecular scissors’ for genome engineering applications. These artificial nucleases offer high specificity coupled with simplicity in design that results from the ability to serially chain transcription activator-like effector repeat arrays to target individual DNA bases. However, these benefits come at the cost of an appreciably large multimeric protein complex, in which DNA cleavage is governed by the nonspecific FokI nuclease domain. Here we report a significant improvement to the standard transcription activator-like effector nuclease architecture by leveraging the partially specific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes. In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptide compact transcription activator-like effector nucleases exhibit overall activity and specificity comparable to currently available designer nucleases. In addition, we harness the catalytic mechanism of I-TevI to generate novel compact transcription activator-like effector nuclease-based nicking enzymes that display a greater than 25-fold increase in relative targeted gene correction efficacy.


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Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding -Trends in Biotechnology

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding -Trends in Biotechnology | TAL effector | Scoop.it

(via T. Lahaye)

Podevin et al, 2013

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding.


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TALEN or Cas9 -- rapid, efficient and specific choices for genomic modifications -Journal of Genetics and Genomics

Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9 mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.


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PLOS Computational Biology: Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PLOS Computational Biology: Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites | TAL effector | Scoop.it
While it had already been discovered that transcription activator-like (TAL) effectors from Xanthomonas pathogens act as transcription factors in the host plant, deciphering the modular code of DNA binding specificity of TAL effectors in 2009 fascinated the scientific community. This modular code opens the possibility to identify virulence targets of natural TAL effectors in host plants including valuable crops. Knowing these targets deepens our understanding of the role of TAL effectors in virulence. At the same time, it is an opportunity to create resistant plants by destroying TAL effector target sites, indispensable for the pathogen, in plant genomes. However, computational methods are needed to effectively scan full genomes or promoteromes for putative target sites. Hence, we propose TALgetter, a new approach for predicting TAL effector target sites. Using TALgetter, we predict target sites of Xanthomonas TAL effectors in the important crop plants rice and sweet orange. Besides novel putative virulence targets of several TAL effectors, we also gain new insights into the biology of TAL effector targeting. The predictions of TALgetter reveal that target sites are preferentially located in the vicinity of the transcription start and that many TAL effectors bind to the TATA-box in the promoters of target genes.
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Plant Genome Engineering with Sequence-Specific Nucleases - Annual Review of Plant Biology

Voytas 2013

 

Recent advances in genome engineering provide newfound control over a plant’s genetic material. It is now possible for most bench scientists to alter DNA in living plant cells in a variety of ways, including introducing specific nucleotide substitutions in a gene that change a protein’s amino acid sequence, deleting genes or chromosomal segments, and inserting foreign DNA at precise genomic locations. Such targeted DNA sequence modifications are enabled by sequence-specific nucleases that create double-strand breaks in the genomic loci to be altered. The repair of the breaks, through either homologous recombination or nonhomologous end joining, can be controlled to achieve the desired sequence modification. Genome engineering promises to advance basic plant research by linking DNA sequences to biological function. Further, genome engineering will enable plants’ biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population.

Expected final online publication date for the Annual Review of Plant Biology Volume 64 is April 29, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

 


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A library of TAL effector nucleases spanning the human genome

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.

 


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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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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. | TAL effector | Scoop.it

via Tom Schreiber, thanks

 

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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Current Opinion in Structural Biology - TAL effectors: function, structure, engineering and applications

Current Opinion in Structural Biology - TAL effectors: function, structure, engineering and applications | TAL effector | Scoop.it

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dromius's curator insight, December 27, 2012 10:32 PM

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
repeat (one of two consecutive polymorphic amino acids that are termed ‘repeat variable diresidues’, or ‘RVDs’) specifies the identity of a single base; collectively the sequential repeats and their RVDs dictate the recognition of sequential bases along one of the two DNA strands. The modular architecture of TAL effectors has facilitated their extremely rapid development and
application as artificial gene targeting reagents, particularly in the form of site-specific nucleases. Recent crystallographic and biochemical analyses of TAL effectors have established the structural basis of their DNA recognition properties and provide clear directions for future research.