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Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins - Nucl. Acids Res.

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins - Nucl. Acids Res. | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Becker et al, 2018

Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins (‘Transcription Activator-Like Effector Dimers’; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

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Efficient enrichment cloning of TAL effector genes from Xanthomonas - MethodsX

Efficient enrichment cloning of TAL effector genes from Xanthomonas - MethodsX | TAL effector science | Scoop.it

(via T. Schreiber)

Tran et al, 2018 Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL effectors in their genomes. Some of the TAL effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL effector genes remains a challenge. In order to isolate complete TAL effector gene repertoires, we developed an enrichment cloning strategy based on

• genome-informed in silico optimization of restriction digestions,
• selective restriction digestion of genomic DNA, and
• size fractionation of DNA fragments.

Our rapid, cheap and powerful method allows efficient cloning of TAL effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.

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TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 ( FAD2) in peanut ( Arachis hypogaea L.) promotes the accumulation of oleic acid

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 ( FAD2) in peanut ( Arachis hypogaea L.) promotes the accumulation of oleic acid | TAL effector science | Scoop.it

(via J. Boch, thx)

Wen et al, 2018

Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5–2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

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Functional and Genome Sequence-Driven Characterization of tal Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Str...

Functional and Genome Sequence-Driven Characterization of tal Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Str... | TAL effector science | Scoop.it

Doucouré et al., 2018

Rice Bacterial Leaf Blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variant

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DNA Nucleases and their Use in Livestock Production

DNA Nucleases and their Use in Livestock Production | TAL effector science | Scoop.it

Petersen, 2018

DNA nucleases, including zinc-finger nucleases (ZFN), transcription activator-like endonucleases (TALENS), and meganucleases, possess long recognition sites and cutting domains and are thus capable of cutting DNA in a very specific manner. These molecular scissors mediate targeted genetic alterations by enhancing the DNA mutation rate via induction of double-strand breaks at a predetermined genomic site. Compared to conventional homologous recombination-based gene targeting, DNA nucleases can increase the targeting rate up to 10,000-fold, and gene disruption via mutagenic DNA repair is stimulated at a similar frequency. The successful application of different DNA nucleases has been demonstrated in a multitude of organisms, including insects, amphibians, plants, nematodes, and mammals, including livestock animals. Recently, another novel class of molecular scissors was described that uses short RNA sequences to target a specific genomic site (Fig. 7.1). The CRISPR/CAS9 originates from a bacterial defense mechanism and can be programmed to target almost any site within a genome. The ease and low costs to create very specific genetic alterations by DNA nucleases have revolutionized the production of genetically modified livestock. Current results indicate that DNA nucleases can be successfully employed in a broad range of organisms which renders them useful for improving the understanding of complex physiological systems, producing genetically modified animals, including creating large animal models for human diseases and creating specific cell lines. Genetic modifications could also increase animal welfare by making dehorning and sexing obsolete or by making farm animals resistant/resilient against specific pathogens. Livestock with a desired phenotype or trait can now be produced with previously unknown precision and ease and within a very short time frame considered to be impossible before their advent. This chapter provides an update on DNA nucleases and their underlying mechanism and focuses on their use in livestock production. It has to be kept in mind that, at the time of writing this chapter, none of the genetically modified livestock has entered the food chain or had been used for the production of livestock-derived products.

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Sequence-specific 5mC detection in live cells based on the TALE-split luciferase complementation system - Analyst (RSC Publishing)

Sequence-specific 5mC detection in live cells based on the TALE-split luciferase complementation system - Analyst (RSC Publishing) | TAL effector science | Scoop.it

Tsuji et al, 2018

We established a method for converting TALE-DNA binding to luminescence, by combining TALE and a sprit luciferase system. Furthermore, using a methylation-sensitive TALE, sequence-specific 5mC detection of genomic DNA was achieved in live cells. This study provides new strategy for exploring the biological functions of 5mC.

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Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes - Biochem Biophys Res C...

Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes - Biochem Biophys Res C... | TAL effector science | Scoop.it

Sun et al, 2018

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a serious bacterial disease that affects citrus trees worldwide. The ectopic expression of TAL effector AvrXa7 in Xcc suppressed canker development. The Xcc strain expressing avrXa7 induced a yellow symptom around the inoculation site. Transcriptome analysis revealed 315 differentially expressed genes, which were categorized into several functional groups. The more interesting genes were those involved in the biosynthesis of terpene and ethylene. In particular, the linoleate 13 S-lipoxygenase gene CsLOX2-1 was found to possess the AvrXa7 binding sequence in the promoter region. The recognition of AvrXa7 to the CsLOX2-1 promoter was subsequently confirmed by yeast one-hybrid and electrophoretic mobility shift experiments. This demonstrated that the TALE effector AvrXa7 promotes CsLOX2-1 expression by directly binding to the promoter sequence. Our findings contribute a valuable clue to identifying the potential genes that can be used to prevent citrus canker.

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A cautionary TALE: how plant breeding may have favoured expanded TALE repertoires in Xanthomonas - Mol. Plant Pathol.

A cautionary TALE: how plant breeding may have favoured expanded TALE repertoires in Xanthomonas - Mol. Plant Pathol. | TAL effector science | Scoop.it

Schandry et al, 2018

Xanthomonas oryzae strains overcome recognition by traditional R genes, Xo1 and Xa1, by deploying structural variants of TALEs that seem to interfere with R gene function, possibly by competitively binding the R protein without eliciting an immune response (Zuluaga et al., 2017). It is conceivable that the loss of TALEs in some US strains is a response to the widespread presence of these R genes in cultivated rice in North America. Consequently, these strains avoid recognition and resistance at the expense of a significant loss of virulence.

We believe that these described counter‐defence mechanisms are a testament to the effect of the deployment of R genes on TALome evolution. Indeed, to date, these mechanisms have been found almost exclusively in X. oryzae strains with expanded TALomes (Fig. 1). We propose that the expansion of TALomes is thus a feature that has been selected for in response to plant resistance, as it allows the bacteria to develop counter‐defence strategies and to quickly adapt to new cultivars.

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Engineering altered protein–DNA recognition specificity - Nucl Acids Res.

Engineering altered protein–DNA recognition specificity - Nucl Acids Res. | TAL effector science | Scoop.it

Bogdanove et al, 2018

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein–protein interactions, modifying protein–DNA interactions is more difficult. This may be related to the structural features of protein–DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein–DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

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Identification of a cell-penetrating peptide applicable to a protein-based transcription activator-like effector expression system for cell engineering - Biomaterials

Takashina et al, 2018

Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11). We named the peptide “nuclear trafficking peptide” (NTP) and applied it to a protein-based artificial transcription factor (NTP-ATF), which was composed of a transcription activator-like effector and transcription domain (VP64). An NTP-ATF designed to the proximal promoter region of the microRNA-302/367 cluster efficiently induced endogenous RNA expression at an extremely low concentration (0.25 nM), and repetitive treatment of mouse embryonic fibroblasts with NTP-ATF generated induced pluripotent stem-like cells, which gave chimeric mice. Together with the observation that recombinant NTP-ATF protein did not induce any apparent cytotoxicity, we propose that NTP-ATF is a promising system for cellular reprogramming applicable to regenerative medicine.

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Recognition of Epigenetic Nucleobases - Phil. Trans. R. Soc. B

Recognition of Epigenetic Nucleobases - Phil. Trans. R. Soc. B | TAL effector science | Scoop.it

Rathi et al, 2018

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.

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The pepper Bs4C proteins are localized to the ER membrane and confer disease resistance to bacterial blight in transgenic rice - Mol.Plant Pathol.

(via T. Schreiber, thx)

Wang et al, 2018

Transcription activator‐like effector (TALE)‐dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced upon infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C‐R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C‐R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C‐R and two other Bs4C‐like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C‐S) and the CaBs4C‐R homolog gene in the cultivar “CM334” of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C‐eCFP fusion proteins were localized to the ER membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter‐Bs4C fusion genes in transgenic rice conferred strain‐specific disease resistance to Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight in rice, and were specifically induced by the Xa10‐incompatible Xoo strain PXO99A(pHM1avrXa10). The results indicated that the Bs4C proteins from the pepper species function broadly in rice and the Bs4C protein‐mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. This article is protected by copyright. All rights reserved.

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Immune modulation in pigs through TALEN-based editing of the RELA locus

Ballantyne 2018

 

Livestock animals are an ancient, vital renewable natural resource. Many livestock
species have the ability to convert inedible crops and waste food into food fit for human
consumption, in the form of meat, eggs and dairy products. As the global demand for
high value animal protein is ever increasing, the livestock market continues to play a
major role in worldwide economics.
Animal disease has the potential to be a huge burden on the livestock industry, impacting both welfare and production. Major outbreaks of transboundary diseases, such as foot and mouth disease, rinderpest and classical swine disease, have resulted in devastating global economic losses. As a result, scientific research is engaged in lowering this impact by generating effective preventative measures and treatments.
One way to reduce livestock disease is to select animals that are genetically
resistant, traditionally carried out through selective animal breeding programs; however, this is a time- consuming process and requires that appropriate genetic variation exists within the population. Advances in genome engineering technologies offer us an alternative approach, with the capability to make genetic improvements in livestock within a single generation. It is hypothesised that resilience to a disease, known as African
swine fever (ASF), could be genetically engineered into the domestic pig.
ASF is a highly contagious disease of domestic pigs and is a re- emerging global threat to the swine industry. It is a lethal haemorrhagic disease caused by a virus, known as the African swine fever virus (ASFV). At present, there is no vaccine or treatment for ASF, and disease control relies on rapid diagnosis, quarantine and the mass slaughter
of animals. Unlike the domestic pig, swine indigenous to Sub-Saharan Africa, such as
the warthog, show no clinical signs of disease following infection with ASFV. A
comparative study was carried out to identify host genetic variation that could underlie
the difference in response to ASFV, with candidate genes selected based on their
potential involvement with the viral protein A238L, involved in immune evasion.
Functional polymorphisms where identified in the porcine RELA gene, encoding
RelA, a subunit of the NF-κB transcription factor family. This evolutionary conserved protein family plays a vital role in mediating inflammatory and immune responses.
The specific RELA polymorphisms identified alter potential phosphorylation sites
within the C-terminal transactivation domain of RelA which have been found to
modulate NF-κB transcriptional activity
in vitro. We set out to investigate whether
genome editing tools could be employed to engineer the RELA sequence of domestic
pigs. Initial attempts targeted the final exon of RELA, producing animals with a
truncatedRelA protein; modified animals lack the final 60 amino acids of the C-terminal transactivation domain. The aim of this thesis was to genotype and characterise the effects of this RELA modification at a molecular, cellular, morphological and whole organism
level. The ultimate goal of this project was to
investigate whether this RELA modification altered the domestic pig’s response to
ASFV in vitro and in vivo. Unlike rela-/
-mice which have an embryonic lethal phenotype, these RELA-edited pigs were born healthy and were fully viable when housed in a typical farm environment. Phenotypic analysis of lymphoid tissues from the RELA-edited pigs demonstrated no significant anatomical or histological changes compared to unmodified counterparts. Pigs homozygous for the RELA mutation had a significantly lower body weight compared to wild-type pigs. Molecular studies of samples from, these pigs have shown that the modified RelA has an altered activity; however, the RELA modified pigs do develop the characteristic disease phenotype when challenged with ASFV. Finally, genome editors have been developed to introduce a specific warthog allele into the domestic pig RELA locus, these editors are currently being
taken forward to produce a novel pig line.
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Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing

Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing | TAL effector science | Scoop.it

Waryah et al, 2018

The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulat

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MitoTALEN reduces mutant mtDNA load and restores tRNA Ala levels in a mouse model of heteroplasmic mtDNA mutation - Nature Medicine

MitoTALEN reduces mutant mtDNA load and restores tRNA Ala levels in a mouse model of heteroplasmic mtDNA mutation - Nature Medicine | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Bacman et al, 2018

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules1. Using a mouse model with a heteroplasmic mtDNA mutation2, we tested whether mitochondrial-targeted TALENs (mitoTALENs)3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNAAla levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.

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Targeted Genome Engineering in Xenopus Using the Transcription Activator-Like Effector Nuclease (TALEN) Technology

Targeted Genome Engineering in Xenopus Using the Transcription Activator-Like Effector Nuclease (TALEN) Technology | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Van NieuwenhuyseN & Vleminckx 2018

Targeted genome engineering technologies are revolutionizing the field of functional genomics and have been extensively used in a variety of model organisms, including X. tropicalis and X. laevis. The original methods based on Zn-finger proteins coupled to endonuclease domains were initially replaced by the more efficient and straightforward transcription activator-like effector nucleases (TALENs), adapted from plant pathogenic Xanthomonas species. Although functional genomics are more recently dominated by the even faster and more convenient CRISPR/Cas9 technology, the use of TALENs may still be preferred in a number of cases. We have successfully implemented this technology in Xenopus and in this chapter we describe our working protocol for targeted genome editing in X. tropicalis using TALENs.

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Fishing for understanding: Unlocking the zebrafish gene editor’s toolbox - Methods

Fishing for understanding: Unlocking the zebrafish gene editor’s toolbox - Methods | TAL effector science | Scoop.it

Simone et al, 2018

The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing. As the zebrafish gene editing toolkit continues to grow, it is becoming increasingly important to understand when and how to utilize which of these technologies for maximum efficacy in a particular project. While CRISPR-Cas9 has brought broad attention to the field of genome engineering in recent years, designer endonucleases have been utilized in genome engineering for more than two decades. This chapter provides a brief overview of designer endonuclease and other gene editing technologies in zebrafish as well as some of their known functional benefits and limitations depending on specific project goals. Finally, selected prospects for additional gene editing tools are presented, promising additional options for directed genomic programming of this versatile animal model system.

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Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs - Theriogenology

Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs - Theriogenology | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Su et al, 2018

Gene editing tools (Zinc-Finger Nucleases, ZFN; Transcription Activator-Like Effector Nucleases, TALEN; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9, CRISPR-Cas9) provide us with a powerful means of performing genetic engineering procedures. A combinational approach that utilizes both somatic cell nuclear transfer (SCNT) and somatic cell gene editing facilitates the generation of genetically engineered animals. However, the associated research has utilized markers and/or selected genes, which constitute a potential threat to biosafety. Microhomologous-mediated end-joining (MMEJ) has showed the utilization of micro-homologous arms (5–25 bp) can mediate exogenous gene insertion. Dairy milk is a major source of nutrition worldwide. However, most people are not capable of optimally utilizing the nutrition in milk because of lactose intolerance. Sulfolobus solfataricus β-glycosidase (LacS) is a lactase derived from the extreme thermophilic archaeon Sulfolobus solfataricus. Our finally aim was to site-specific integrated LacS gene into cow's genome through TALEN-mediated MMEJ and produce low-lactose cow. Firstly, we constructed TALENs vectors which target to the cow's β-casein locus and LacS gene expression vector which contain TALEN reorganization sequence and micro-homologous arms. Then we co-transfected these vectors into fetal derived skin fibroblasts and cultured as monoclone. Positive cell clones were screened using 3′ junction PCR amplification and sequencing analysis. The positive cells were used as donors for SCNT and embryo transfer (ET). Lastly, we detected the genotype through PCR of blood genomic DNA. This resulted in a LacS knock-in rate of 0.8% in TALEN-treated cattle fetal fibroblasts. The blastocyst rate of SCNT embryo was 27%. The 3 months pregnancy rate was 20%. Finally, we obtained 1 newborn cow (5%) and verified its genotype. We obtained 1 site-specific marker-free LacS transgenic cow. It provides a basis to solve lactose intolerance by gene engineering breeding. This study also provides us with a new strategy to facilitate gene knock-ins in livestock using techniques that exhibit improved biosafety and intuitive methodologies.

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Functional and genome sequence-driven characterization of tal effector gene repertoires reveals novel variants with altered specificities in closely related Malian Xanthomonas oryzae pv. oryzae str...

(Via T. Schreiber, thx!)

Doucoure et al, 2018

Rice Bacterial Leaf Blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

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TALE - Mediated Inhibition of Replication of Begomoviruses - Int. J. Agr. Biol.

(via T. Lahaye, thx)

Khan et al, 2018

During the last decade, unprecedented progress in the field of genome modification has been witnessed with various applications in basic and applied biology. Genome editing with specific DNA binding proteins has shown higher specificity and fidelity. Artificially engineered proteins such as zinc fingers (ZFs), transcription activator-like effectors (TALEs) and clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nucleases (e.g., Cas9) have been used to edit genomes of several plants species such as wheat, rice, soybean, potato, tomato, tobacco, Arabidopsis etc. Engineered proteins with nuclease domain, ZFNs, TALENs, CRISPR/Cas9, can be used to induce double-strand breaks (DSB) in the target genomes. In eukaryotic systems, double strand breaks are repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR) based repair mechanisms resulting in knockdown or malfunction of the targeted gene. Begomoviruses are becoming a serious threat to a number of crops in Pakistan. The present study was initiated with the objective to demonstrate suppression of replication of cotton leaf curl virus (CLCuV) using TALE technology. The most conserved DNA sequence of begomoviruses, nonanucleotide, was targeted to achieve a broad-spectrum resistance against CLCuV prevalent in Pakistan. Activity of TALEs for virus suppression was successfully demonstrated in Nicotiana benthamiana by challenging with infectious clones of cotton leaf curl Kokhran virus (CLCuKV). Virus accumulation was determined by qPCR. The plants showed varying degrees of resistance to CLCuKV in three ways; attenuated virus infection, delayed symptoms and lower virus titer. Our results successfully demonstrated the potential of TALE technology for CLCuV suppression and offer a broader genome targeting platform for suppression of other viruses.

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Conformational heterogeneity allows access to DNA in longer Transcription Activator-Like Effector (TALE) arrays. - BioRxiv

Geiger-Schuller et al, 2018

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. Here, we examine the kinetics of DNA binding for a set of TALE arrays with identical repeats of varying length (and repeat number) using single molecule microscopy. Using a new deterministic modeling approach to test kinetic models consistent with data, we find evidence for conformational heterogeneity in both the free- and DNA-bound TALE arrays. We connect these results with previous work demonstrating populations of partly folded TALE states. TALEs forming less than one superhelical turn around DNA access partly folded open states that inhibit DNA binding, whereas TALEs forming more than one turn access partly folded open states that facilitate DNA binding. Overall, we find that increasing repeat number results in significantly slower interconversion between and among DNA-free and DNA-bound states. These findings highlight the role conformational dynamics can play in facilitating the assembly of large complexes.

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Xanthomonas oryzae pv. oryzae TALE proteins recruit OsTFIIAγ1 to compensate for the absence of OsTFIIAγ5 in bacterial blight in rice - Mol Plant Pathol.

Ma et al, 2018

Xanthomonas oryzae pv. oryzae (Xoo), causal agent of bacterial blight (BB) of rice, uses transcription activator‐like effectors (TALEs) to interact with the basal transcription factor gama subunit OsTFIIAγ5 (Xa5) and activates transcription of host genes. However, how OsTFIIAγ1, the other OsTFIIAγ protein, functions in the presence of TALEs remains unclear. In this study, we show that OsTFIIAγ1 plays a compensatory role in the absence of Xa5. The expression of OsTFIIAγ1, which is activated by TALE PthXo7, increased the expression of host genes targeted by avirulent and virulent TALEs. Defective OsTFIIAγ1 rice lines showed reduced expression of the TALE‐targeted susceptibility (S) genes, OsSWEET11 and OsSWEET14, which resulted in increased BB resistance. Selected TALEs (PthXo1, AvrXa7, and AvrXa27) were evaluated for interactions with OsTFIIAγ1, Xa5 and xa5 (naturally‐occurring mutant form of Xa5) using biomolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST). BiFC and MST demonstrated that the three TALEs bind Xa5 and OsTFIIAγ1 with a stronger affinity than xa5. These results provide insight into the complex roles of OsTFIIAγ1 and OsTFIIAγ5 in TALE‐mediated host gene transcription. This article is protected by copyright. All rights reserved.

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Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal TALE-Binders - JACS

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal TALE-Binders - JACS | TAL effector science | Scoop.it

Giess et al, 2018

5-methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridiza-tion probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epi-genetic nucleobase selectivity. However, only partial selectivi-ties for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We here overcome this limitation by destroying and re-inducing nucleobase selectivity in TA-LEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach now enables the fully selective detection of each oxidized 5mC in complex DNA samples by programmable probes.

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Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire - New Phytol.

(via T. Schreiber)

Denance et al, 2018

  • Xanthomonas transcription activator‐like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE‐encoding (tal) genes were thought to be absent from Brassicaceae‐infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences.
  • We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near‐complete description of the TALEs found in Xc (Xc TALome).
  • The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis.
  • This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE‐mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.
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Functional characterization of the citrus canker susceptibility gene CsLOB1 - Mol. Plant Pathol.

(via T. Schreiber, thx)

Duan et al, 2018

Xanthomonas citri ssp. citri (Xcc) is an important plant‐pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator‐like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1‐GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX‐treated wild‐type plants and in non‐treated CsLOB1‐GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1‐GR transgenic plants following DEX treatment. When CsLOB1‐GR‐transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX‐treated transgenic plant leaves, but not on mock‐treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX‐induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4‐type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

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TALEN-Based Knockout System

TALEN-Based Knockout System | TAL effector science | Scoop.it

Yoshida & Treen, 2018

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

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