TAL effector science
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TAL effector science
infos on novel DNA-binding proteins of bacteria and their biotech use
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Off-target effects of engineered nucleases - Yee - 2016 - The FEBS Journal

Jee, 2016

Recent advances in gene editing with engineered nucleases have transformed our ability to manipulate the genome from diverse organisms for applications ranging from biomedical research to disease treatment. A major complication with these engineered nucleases is the binding of the nuclease to unintended genomic sites that share sequence homology with the on-target site. Cleavage of these off-target sites followed by DNA repair using normal cellular DNA repair mechanisms can cause gene mutation or gross chromosome rearrangement. Identification of nuclease-generated off-target sites is a daunting task due to the size and complexity of the mammalian genome. Five unbiased, genome –wide strategies have been developed to detect the off-target cleavage. Some of these strategies reach the sensitivity near the detection limit of directed deep sequencing and have sufficient precision and resolution to objectively assessing the off-target effect of any engineered nuclease. Significant progress has also been made recently to boost the nuclease targeting specificity by protein engineering to modify the structure of the nuclease and alter the interaction with its genomic target. In several studied cases, the off-target effect generated by the modified nuclease is completely eliminated. These modified nucleases significantly improve the overall fidelity of gene editing. These developments will enable gene editing tools to be applied more broadly and safely in basic research and disease treatment.

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Engineered Swine Models of Cancer - Frontiers in Cancer Genetics

Watson et al, 2016

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

dromius's insight:

"Because the RNA-guided site-specific platforms (e.g., CRISPR-based) may allow U-G base-pairing, their fidelity may be lower than the protein-based platforms (e.g., TALENs). Consequently, although we have used most of the site-specific nuclease platforms, for fidelity and efficiency, we find TALEN-induced cleavages are the best balance for reliable gene-editing"

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Spatial organization of heterologous metabolic system in vivo based on TALE

Spatial organization of heterologous metabolic system in vivo based on TALE | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Zhu et al, 2016

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

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TALEN gene editing takes aim on HIV - Springer

TALEN gene editing takes aim on HIV - Springer | TAL effector science | Scoop.it

Benjamin et al, 2016

Transcription activator-like effector nucleases (TALENs) are one of several types of programmable, engineered nucleases that bind and cleave specific DNA sequences. Cellular machinery repairs the cleaved DNA by introducing indels. In this review, we emphasize the potential, explore progress, and identify challenges in using TALENs as a therapeutic tool to treat HIV infection. TALENs have less off-target editing and can be more effective at tolerating HIV escape mutations than CRISPR/Cas-9. Scientists have explored TALEN-mediated editing of host genes such as viral entry receptors (CCR5 and CXCR4) and a protein involved in proviral integration (LEDGF/p75). Viral targets include the proviral DNA, particularly focused on the long terminal repeats. Major challenges with translating gene therapy from bench to bedside are improving cleavage efficiency and delivery, while minimizing off-target editing, cytotoxicity, and immunogenicity. However, rapid improvements in TALEN technology are enhancing cleavage efficiency and specificity. Therapeutic testing in animal models of HIV infection will help determine whether TALENs are a viable HIV treatment therapy. TALENs or other engineered nucleases could shift the therapeutic paradigm from life-long antiretroviral therapy toward eradication of HIV infection.

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Gene Editing Market (Crispr, Talen, ZFN, Antisense technology) Forecasts to 2024 - news.sys-con.com

Gene editing has generated a lot of excitement in academia and drug development. It promise is two-fold: the unique ability to correct genetic mutations that may cause disease; and its utility in creating and controlling genetic information within patient cells. Emerging science suggests that permanently fixing or "editing" mutated cells, or creating safer and more potent cell-based products with this technology could provide curative, one-time treatments for patients suffering from a broad range of diseases.

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Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice - Gene Therapy

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Chapdelaine et al, 2016

Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

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Screening Strategies for TALEN-Mediated Gene Disruption - Springer

Screening Strategies for TALEN-Mediated Gene Disruption - Springer | TAL effector science | Scoop.it

Boris Reljić & David A. Stroud, 2016

Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.

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Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies - Scientific Reports

Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies - Scientific Reports | TAL effector science | Scoop.it

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Itokawa et al, 2016

Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

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TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells - Scientific Rep.

TALENs-directed knockout of the full-length transcription factor Nrf1α that represses malignant behaviour of human hepatocellular carcinoma (HepG2) cells - Scientific Rep. | TAL effector science | Scoop.it

Ren et al, 2016

The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α−/− cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α−/− cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α−/− cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration).

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Genome Engineering with TALE and CRISPR Systems in Neuroscience - Frontiers in Neurogenomics

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Lee et al, 2016

Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.

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Untested ‘designer cell’ treatment saved baby dying from leukemia - Joliet Daily Science

Untested ‘designer cell’ treatment saved baby dying from leukemia - Joliet Daily Science | TAL effector science | Scoop.it
A one-year-old girl baby was suffering from leukemia has been cleared of the cancer after becoming the first human in the world to try a new, untested ‘designer cell’ treatment that creates designer immune cells. Now the scientists must run further clinical trials to test if the TALEN technology can be used on a larger …
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Editorial Prerogative and the Plant Genome - J. Genet. Genomics

Voytas 2016

In the coming decades, plant agriculture must produce the food needed to sustain the world’s burgeoning population. This challenge is made more daunting in light of a changing climate, scarcity of inputs such as water and fertilizer, and the need to grow more crops on increasingly marginal land. To achieve food security, we will need to fully exploit plant genetics to develop new crop varieties that produce higher yields of healthier food. Crop improvement through breeding has been the foundation of modern agriculture, resulting in steady increases in productivity of the major commodity crops (Sleper and Poehlman, 2006). Additional genetic variation of value has been created through mutagenesis strategies (Micke et al., 1990). More recently, gene addition through transgenesis has provided valuable traits that impact yield, including herbicide tolerance and resistance to insect pests (Gepts, 2002). Now, with the advent of genome editing, we can directly alter the genetic blueprints of crop species – a powerful approach to create genetic variation needed to produce abundant, healthy food.

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Sequence-Specific Nucleases for Genetic Improvement of Potato - Amer. J. Potato Res.

Sequence-Specific Nucleases for Genetic Improvement of Potato - Amer. J. Potato Res. | TAL effector science | Scoop.it

Butler & Douches 2016

Genome editing using sequence-specific nucleases (SSNs) is rapidly becoming a standard tool for genetic engineering in crop species. The implementation of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) for inducing double-strand breaks enables targeting of virtually any sequence for genetic modification. Targeted mutagenesis via nonhomologous end-joining (NHEJ) and gene targeting via homologous recombination (HR) have been demonstrated in a number of plant species but reports have been limited in vegetatively propagated crops, such as potato (Solanum tuberosum Group Tuberosum L.). This review provides a historical overview of genetic engineering in agriculture, applications of SSN technologies for genome editing in plant species, and prospects of using SSNs for genetic improvement of potato.

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Production of hornless dairy cattle from genome-edited cell lines - Nature Biotechnology

Production of hornless dairy cattle from genome-edited cell lines - Nature Biotechnology | TAL effector science | Scoop.it

Carlson et al, 2016

Physical dehorning of dairy cattle is practiced to protect animals and their handlers. Genetic analyses have identified variants that are associated with hornlessness (referred to as 'polled') in cattle, a trait that is common in beef but rare in dairy breeds. We have introgressed a candidate POLLED allele into dairy cattle by genome editing and reproductive cloning, providing both evidence for genetic causation and a means to introduce POLLED into livestock with the potential to improve the welfare of millions of cattle annually.

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A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL
effectors with diverse predicted targets and is effective against African strains of Xanthomonas o...

A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL <br/>effectors with diverse predicted targets and is effective against African strains of Xanthomonas o... | TAL effector science | Scoop.it

(Via T. Lahaye, thx)

Triplett et al, 2016

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable diresidues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes.

dromius's insight:

This is cool! Evidence for a Bs4-type recognition in rice. Bs4 is a TIR-NB-LRR, unlikely to be present in monocot rice. Mechanism behind NB-LRR recognition of diverse TALs is still not solved. Can't wait to hear what gene in the 1 Mb target interval confers this resistance.

 

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Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA - PLOS One

Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA - PLOS One | TAL effector science | Scoop.it

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Stoddard et al, 2016

Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the Nicotiana benthamiana ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were 10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from Arabidopsis thaliana genes to the TALEN coding sequence. UTRs from an A. thaliana adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.

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Attitudes towards Governance of Gene Editing - Asian Biotechnology Development Review | RIS

Attitudes towards Governance of Gene Editing - Asian Biotechnology Development Review | RIS | TAL effector science | Scoop.it

Kuzma et al, 2016

Gene editing technologies are revolutionising plant biotechnology.
They allow for the rapid editing of multiple genes with either mutational,cisgenic, or transgenic approaches. They are also challenging regulations in several countries, as definitions and processes are based upon first generation
methods for genetic engineering. In this paper, we describe results from a U.S. study investigating the attitudes of subject matter experts (SMEs) towards the governance of genome editing. We find some areas where SMEs seem to agree, including the need for pre-market oversight and stakeholder engagement.
However, the SMEs had different visions as to the novelty of the technology, primary issues of concern, hopes for the technology, and what specifically should be done in a regulatory context. Key narratives arose including the view that gene editing provides an new opportunity to rethink the oversight of agricultural biotechnology to improve existing systems (adapter view), that
gene editing although revolutionary should undergo less regulation than 1st generation biotechnology (technohype-hyporeg view), and that gene editing makes the engineering process so easy that risk analysis and the regulatory system might not be able to accommodate the speed of development and thus greater caution is warranted (systems view). Current policy debates are revealing these differing perspectives,and formal decisions about how to govern this next generation of agricultural biotechnology are pending.

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Engineered Swine Models of Cancer - Frontiers in Cancer Genetics

(via T. Schreiber, thx)

Watson et al, 2016

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

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Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa -PNAS

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Shinkuma et al, 2016

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

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Biochemical characterization of modular DNA-binding domains of novel TALE-like proteins - Dissertation

Wolf, 2016

Members of the plant pathogenic bacterial genus Xanthomonas inject TALEs (Transcription Activator Like Effector) by a type III secretion system into host plant cells. Inside the plant cells TALEs bind and activate host genes thereby promoting bacterial disease. The DNA binding domain of TALEs is modular and consists of imperfect 33-35 long tandem-arranged amino acid repeats. Each repeat binds to a single nucleotide with position 13, determining base specificity. The base specificity of distinct residues in position 13 is known as the TALE code. This TALE code provides the possibility to create custom TALEs with desired DNA target specificity or to predict DNA targets of native TALEs. This work characterizes two new members of the TALEs, called TALE-likes: (1) Bats, which derive from the bacterium Burkholderia rhizoxinica and (2) MOrTLs, whose DNA sequences were found in a marine metagenomics database. We demonstrate that DNA binding preferences of these two classes of TALE-likes can be predicted with the TALE-code. Yet, some of the repeats have a lower base specificity than TALEs. Additionally the TALE-likes have a different affinity to DNA and higher protein stability compared to TALEs. Analysis of protein chimeras showed that repeats of TALEs and TALE-like proteins are compatible and can be used to create protein chimeras. The TALE-likes have different DNA affinities and protein stabilities as compared to the TALEs. They can be adapted to create new proteins and protein chimeras with new useful properties.

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A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells - PLOS One

A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells - PLOS One | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Findlay et al, 2016

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs “mismatch nucleases” T7E1 or “Surveyor” that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an “all-in-one” CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

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TALEN-Mediated Inactivation of PD-1 in Tumor-Reactive Lymphocytes Promotes Intratumoral T-cell Persistence and Rejection of Established Tumors - Cancer Research

Menger er al., 2016


This proof-of-concept study demonstrates that advanced adoptive T-cell therapies for cancer can be enhanced by genomic editing strategies to bypass immune checkpoints.

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Libraries of Synthetic TALE-Activated Promoters: Methods and Applications - Methods Enzymol.

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications - Methods Enzymol. | TAL effector science | Scoop.it

Schreiber & Tissier 2016

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits.

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Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants - Plant Physiol.

Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants - Plant Physiol. | TAL effector science | Scoop.it

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 Sauer et al, 2016

Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5′-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.

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TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change And Herbicide-Resistant Rice - J. Genet. Genomics

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change And Herbicide-Resistant Rice - J. Genet. Genomics | TAL effector science | Scoop.it

Li et al, 2016

Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence specific endonucleases, especially zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end joining (NHEJ) repair to the DNA double-strand breaks (DSB) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4% to 6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

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