TALE effector Design and Delivery
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TALE effector Design and Delivery
Topics related to the Trans Activator Like Effector technologies application and delivery
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An efficient TALEN mutagenesis system in rice - Methods

An efficient TALEN mutagenesis system in rice - Methods | TALE effector Design and Delivery | Scoop.it

Chen et al, 2014

Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4–5 months.


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Comprehensive analysis of the specificity of transcription activator-like effector nucleases - Nucl. Acids. Res.

Comprehensive analysis of the specificity of transcription activator-like effector nucleases - Nucl. Acids. Res. | TALE effector Design and Delivery | Scoop.it

(via T. Lahaye, thx)

Juillerat et al, 2014

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.


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Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells - Methods

Holkers et al, 2014

Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 “safe harbor” locus on human chromosome 19.


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PLOS ONE: Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

PLOS ONE: Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus | TALE effector Design and Delivery | Scoop.it
PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
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Helper-dependent adenoviral liver gene therapy protects against ...

Helper-dependent adenoviral liver gene therapy protects against ... | TALE effector Design and Delivery | Scoop.it
Helper-dependent adenoviral liver gene therapy protects against induced attacks and corrects protein folding stress in acute intermittent porphyria mice. Carmen Unzu1,; Ana Sampedro1,; Itsaso Mauleón1,; Manuela ...
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Week 13, constructing logic circuits in mammalian cells | A geek with ...

Week 13, constructing logic circuits in mammalian cells | A geek with ... | TALE effector Design and Delivery | Scoop.it
To make a simple OR gate they place two binding sites for different TALE repressors before the constitutive promoter followed by an expressor gene. To turn it into a NOR gate, they added another binding site next to the other ...
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TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome - PNAS

TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome - PNAS | TALE effector Design and Delivery | Scoop.it

(via T. Lahaye, thx)

Ochiai et al, 2014

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease–mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.


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TALE-mediated modulation of transcriptional enhancers in vivo - Nature Methods

TALE-mediated modulation of transcriptional enhancers in vivo - Nature Methods | TALE effector Design and Delivery | Scoop.it

Crocker & Stern 2013

We tested whether transcription activator–like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.


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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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Scientists develop an engineered cardiac tissue model to study the human heart - EurekAlert (press release)

Scientists develop an engineered cardiac tissue model to study the human heart
EurekAlert (press release)
They also found that these human engineered heart tissues were able to incorporate new genetic information carried by adenovirus.
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Ronald Crystal, M.D., receives Pioneer Award - Phys.Org

Ronald Crystal, M.D., receives Pioneer Award - Phys.Org | TALE effector Design and Delivery | Scoop.it
Ronald Crystal, M.D., receives Pioneer Award
Phys.Org
In recognition of his seminal work on adenoviral vectors, which accelerated the translation of gene therapy from the research laboratory to the clinic, Ronald G.
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Does the body's immune response to viral vector delivery systems affect the safety and efficacy of GT-Science Codex

Does the body's immune response to viral vector delivery systems affect the safety and efficacy of GT-Science Codex | TALE effector Design and Delivery | Scoop.it

Science Codex
Does the body's immune response to viral vector delivery systems affect the saftey and efficacy of gene therapy?

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TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting - PLOS One

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting - PLOS One | TALE effector Design and Delivery | Scoop.it

(via T. Lahaye, thx)

Yanik et al, 2013

Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.


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Designing and Testing the Activities of TAL Effector Nucleases - Methods Mol. Biol.

Designing and Testing the Activities of TAL Effector Nucleases - Methods Mol. Biol. | TALE effector Design and Delivery | Scoop.it

Lin et al, 2013

Transcription activator-like effector nucleases (TALENs) have rapidly developed into a powerful tool for genome editing. To avoid labor-intensive and time-consuming experimental screening for active TALENs, a scoring system can help select optimal target sites. Here we describe a procedure to design active TALENs using a scoring system named Scoring Algorithm for Predicted TALEN Activity (SAPTA) and a method to test the activity of individual and pairs of TALENs.


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Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator-Like Effector Nucleases (TALENS) into Mouse Zygotes - Mol. Cell. Biol.

Li et al, 2014

Injection of TALEN mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescent visualization of phosphorylated histone-2A (γH2AX) combined with fluorescence in situ hybridization (FISH) revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within six hours of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB X C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to “spacer” sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic co-injection of TALEN mRNAs directed to Agouti, miR-205, and the Arf tumor suppressor locus yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knock-out technology in embryonic stem cells.


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Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene - PLOS Pathogens

Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene  - PLOS Pathogens | TALE effector Design and Delivery | Scoop.it

(via T. Lahaye, thx)

Cernadas et al, 2014

Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.


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Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering - PLOS One

Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering - PLOS One | TALE effector Design and Delivery | Scoop.it

(via T. Lahaye, thx)

Liu et al, 2014

Delivery of TAL effector nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

 

 


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A novel helper-dependent adenovirus-based cell culture model for ...

A novel helper-dependent adenovirus-based cell culture model for ... | TALE effector Design and Delivery | Scoop.it
A novel helper-dependent adenovirus-based cell culture model for Hepatitis C virus replication and production | Health4News. By health4infos on August 30, 2013. By using the hepatitis C virus (HCV) genotype 2a JFH-1 or its chimeric strains, ...
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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 | TALE effector Design and Delivery | 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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Adeno-associated virus type 2 preferentially integrates single genome copies ... - 7thSpace Interactive (press release)

Adeno-associated virus type 2 preferentially integrates single genome copies ...
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Rapid Assembly of Customized TALENs into Multiple Delivery Systems - PLOS One

Rapid Assembly of Customized TALENs into Multiple Delivery Systems - PLOS One | TALE effector Design and Delivery | Scoop.it

(via T. Schreiber, thx)

Zhang et al, 2013

Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated “user friendly” TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains.


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Gene Therapy's Next Generation - Bio-IT World

Gene Therapy's Next Generation - Bio-IT World | TALE effector Design and Delivery | Scoop.it
Gene Therapy's Next Generation
Bio-IT World
Zhang, whose academic lab is part of the Broad Institute of MIT and Harvard, has years of experience working with both ZFNs and the similar TALEN* system.
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Biologists make first mouse model for MERS - Nature.com

Biologists make first mouse model for MERS - Nature.com | TALE effector Design and Delivery | Scoop.it
Biologists make first mouse model for MERS Nature.com To create a more practical model, Stanley Perlman, a microbiologist of the University of Iowa in Iowa City, and his colleagues put the gene encoding human DPP4 into an adenovirus to which mice...
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First monkeys with customized mutations born

First monkeys with customized mutations born | TALE effector Design and Delivery | Scoop.it
Milestone for targeted gene-editing technology promises better models for human diseases.
Samantha Nicholson's insight:

Milestone for engineering models for human disease

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Art of Cell Culture by National Geographic

Art of Cell Culture by National Geographic | TALE effector Design and Delivery | Scoop.it
National Geographic
Phenomena:
National Geographic
This is the second edition of Lab Culture, a photo- and video-heavy series on the culture and methods of different labs.

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