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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Efficient genome engineering of Toxoplasma gondii using the TALEN technique - Parasites & Vectors

Efficient genome engineering of Toxoplasma gondii using the TALEN technique - Parasites & Vectors | TAL effector science | Scoop.it

Chen et al, 2019

Transgenic Toxoplasma gondii was successfully generated by the TALEN system. The eGFP-tagged AAH2 could be detected by in vivo imaging.
This study verified the feasibility of using TALEN technology for T. gondii research and provided an in vivo imaging method for in vivo research of bradyzoite-stage proteins.

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Transcriptional Reprogramming of Rice Cells by Xanthomonas oryzae TALEs - Frontiers Plant Sci.

Transcriptional Reprogramming of Rice Cells by Xanthomonas oryzae TALEs - Frontiers Plant Sci. | TAL effector science | Scoop.it

Muecke et al, 2019

Rice-pathogenic Xanthomonas oryzae bacteria cause severe harvest loss and challenge a stable food supply. The pathogen virulence relies strongly on bacterial TALE (transcription activator-like effector) proteins that function as transcriptional activators inside the plant cell. To understand the plant targets of TALEs, we determined the genome sequences of the Indian X. oryzae pv. oryzae (Xoo) type strain ICMP 3125T and the strain PXO142 from the Philippines. Their complete TALE repertoire was analyzed and genome-wide TALE targets in rice were characterized. Integrating computational target predictions and rice transcriptomics data, we were able to verify 12 specifically induced target rice genes. The TALEs of the Xoo strains were reconstructed and expressed in a TALE-free Xoo strain to attribute specific induced genes to individual TALEs. Using reporter assays, we could show that individual TALEs act directly on their target promoters. In particular, we show that TALE classes assigned by AnnoTALE reflect common target genes, and that TALE classes of Xoo and the related pathogen X. oryzae pv. oryzicola share more common target genes than previously believed. Taken together, we establish a detailed picture of TALE-induced plant processes that significantly expands our understanding of X. oryzae virulence strategies and will facilitate the development of novel resistances to overcome this important rice disease.

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Design, Construction, and Application of Transcription Activation-Like Effectors - in: Viral Vectors for Gene Therapy - Methods and Protocols | Fredric P. Manfredsson | Springer

Design, Construction, and Application of Transcription Activation-Like Effectors - in: Viral Vectors for Gene Therapy - Methods and Protocols | Fredric P. Manfredsson | Springer | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Deng et al, 2019

Transcription activator-like effectors (TALEs) are modular proteins derived from the plant Xanthomonas sp. pathogen that can be designed to target unique DNA sequences following a simple cipher. Customized TALE proteins can be used in a variety of molecular applications that include gene editing and transcriptional modulation. Presently, we provide a brief primer on the design and construction of TALEs. TALE proteins can be fused to a variety of different effector domains that alter the function of the TALE upon binding. This flexibility of TALE design and downstream effect may offer therapeutic applications that are
discussed in this section. Finally, we provide a future perspective on TALE technology and what challenges remain for successful translation of gene-editing strategies to the clinic.

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Analysis of genetic diversity of Xanthomonas oryzae pv. oryzae populations in Taiwan - Sci. Reports

Analysis of genetic diversity of Xanthomonas oryzae pv. oryzae populations in Taiwan - Sci. Reports | TAL effector science | Scoop.it

(via T. Schreiber, thx.)

Chien et al, 2019

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major rice disease. In Taiwan, the tropical indica type of Oryza sativa originally grown in this area is mix-cultivated with the temperate japonica type of O. sativa, and this might have led to adaptive changes of both rice host and Xoo isolates. In order to better understand how Xoo adapts to this unique environment, we collected and analyzed fifty-one Xoo isolates in Taiwan. Three different genetic marker systems consistently identified five groups. Among these groups, two of them had unique sequences in the last acquired ten spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) region, and the other two had sequences that were similar to the Japanese isolate MAFF311018 and the Philippines isolate PXO563, respectively. The genomes of two Taiwanese isolates with unique CRISPR sequence features, XF89b and XM9, were further completely sequenced. Comparison of the genome sequences suggested that XF89b is phylogenetically close to MAFF311018, and XM9 is close to PXO563. Here, documentation of the diversity of groups of Xoo in Taiwan provides evidence of the populations from different sources and hitherto missing information regarding distribution of Xoo populations in East Asia.

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TALEN-mediated gene-editing of HBG in human hematopoietic stem cells leads to therapeutic fetal hemoglobin induction - Mol. Therapy

TALEN-mediated gene-editing of HBG in human hematopoietic stem cells leads to therapeutic fetal hemoglobin induction - Mol. Therapy | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Lux et al, 2019

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.

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Establishment of a conditional TALEN system using the translational enhancer dMac3 and an inducible promoter activated by glucocorticoid treatment to increase the frequency of targeted mutagenesis ...

Establishment of a conditional TALEN system using the translational enhancer dMac3 and an inducible promoter activated by glucocorticoid treatment to increase the frequency of targeted mutagenesis ... | TAL effector science | Scoop.it

Onodera et al, 2018

Transcription activator-like effector nuclease (TALEN) is an artificial nuclease that causes DNA cleavage at the target site and induces few off-target reactions because of its high sequence specificity. Powerful and variable tools using TALENs can be used in practical applications and may facilitate the molecular breeding of many plant species. We have developed a convenient construction system for a plant TALEN vector named the Emerald Gateway TALEN system. In this study, we added new properties to this system, which led to an increase in the efficiency of targeted mutagenesis. Rice dMac3 is a translational enhancer that highly increases the efficiency of translation of the downstream ORF. We inserted dMac3 into the 5' untranslated region of the TALEN gene. In the cultured rice cells to which the TALEN gene was introduced, the frequency of targeted mutagenesis was highly increased compared with those altered using the conventional system. Next, the promoter for the TALEN gene was replaced with iPromoter, and its expression was stringently controlled by a GVG transcription factor that was activated in the presence of glucocorticoid. This conditional expression system worked effectively and led to a higher frequency of targeted mutagenesis than that by the constitutive expression system, while no mutagenesis was detected without glucocorticoid treatment. These results suggest that our system can be applied to genome editing to create the desired mutation.

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AvrBs3-like genes and TAL Effectors Specific to Race Structure in Xanthomonas axonopodis pv. glycines - Thai J. Agric. Sci.

Kladsuwan et al, 2017

The avrBs3-like genes designated avrXg3 and avrXg2 cloning from representative strains of Xanthomonas axonopodis pv. glycines (Xag), a causal agent of bacterial pustule disease were harbored by race 1 (new strain SP4); and race 2 (recent strain 12-2) respectively, whereas avrXg1 (reported by Athinuwat et al., 2009) was uniquely detected in race 3 (recent strain KU-P-SW005) that characterized based on their ability to confer virulence toward soybean cultivars. Most of the new strains collected in this study (169 strains) belonged to race 2 (62.72%), the highly virulence strain group. Structural analysis of transcription activator-like effector (TALe) genes determined to particular races was shown that these race strains including race 1 strain SP4, race 2 strain 12-2, and race 3 strain KU-P-SW005 carried TAL effectors of 6 (tal1a, tal1b, tal1c, tal2a, tal2b, and tal3), 6 (same as strain SP4), and 5 (same as strain SP4 but tal1c absent) tale gene members that guided by 15-23, 15-23, and 15-20 repeat variable diresidues (RVDs) respectively. The individual mutants of each tal genes (17-tal derivatives) revealed that only tal2b in each race strains (XagSP4/tal2b, Xag12-2/ tal2b, XagKU-P-SW005/tal2b) exhibited a positive regulator of similar defense-related phenotype on soybean cultivars of Xag/avrBs3-race typing group, suggesting R genes mediated specifically recognition of directly tal2b induction. Phylogenetic construction and hybridization analysis demonstrated high potential in rapid genotyping of many Xag-race strains using primers and DNA probes designed from either specific avrBs3 or tale-like genes characterized. Genetic mapping and RT-PCR evidence showed that soybean GmLOB1 resistance genes mediated recognition of Xag12-2/tal2b-expressing strain (highly virulent strain) to give disease development on resistance cultivar, Williams82. The tal2b and non-TALe type of type III effectors that may establish a co-opted activity targeting host R genes other than GmLOB1 should be experimentally-further analyzed. This is the first description of TALes indicating their translocation through the Xag-T3SS which the work reports two new avrBs3-like genes and seventeen tale- like genes characterized from soybean-3 pathogenic Xag race strains.

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Polarized displacement by transcription activator-like effectors for regulatory circuits - Nature Chem. Biol.

Polarized displacement by transcription activator-like effectors for regulatory circuits - Nature Chem. Biol. | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Lebar et al, 2018

The interplay between DNA-binding proteins plays an important role in transcriptional regulation and could increase the precision and complexity of designed regulatory circuits. Here we show that a transcription activator-like effector (TALE) can displace another TALE protein from DNA in a highly polarized manner, displacing only the 3′- but not 5′-bound overlapping or adjacent TALE. We propose that the polarized displacement by TALEs is based on its multipartite nature of binding to DNA. The polarized TALE displacement provides strategies for the specific regulation of gene expression, for construction of all two-input Boolean genetic logic circuits based on the robust propagation of the displacement across multiple neighboring sites, for displacement of zinc finger-based transcription factors and for suppression of Cas9–gRNA-mediated genome cleavage, enriching the synthetic biology toolbox and contributing to the understanding of the underlying principles of the facilitated displacement.

 

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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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Functional instability allows access to DNA in longer Transcription Activator-Like Effector (TALE) arrays - eLIFE

Functional instability allows access to DNA in longer Transcription Activator-Like Effector (TALE) arrays - eLIFE | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Geiger-Schuller et al, 2019

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. How TALE repeat domains wrap around DNA, often extending more than 1.5 helical turns, without using external energy is not well understood. Here, we examine the kinetics of DNA binding of TALE arrays with varying numbers of identical repeats. Single molecule fluorescence analysis and deterministic modeling reveal conformational heterogeneity in both the free- and DNA-bound TALE arrays. Our findings, combined with previously identified partly folded states, indicate a TALE instability that is functionally important for DNA binding. For TALEs forming less than one superhelical turn around DNA, partly folded states inhibit DNA binding. In contrast, for TALEs forming more than one turn, partly folded states facilitate DNA binding, demonstrating a mode of 'functional instability' that facilitates macromolecular assembly. Increasing repeat number slows down interconversion between the various DNA-free and DNA-bound states.

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Genome Editing: Tools and Application in Plants - OAJMB

Gudeta, 2019

The emergence of genome editing methods promises a real revolution in genetic engineering. These technologies rely on engineered nucleases that cleave DNA in a sequence-specific manner because of the presence of a sequence-specific DNA-binding domain or RNA sequence. Genome editing by engineered nuclease have the potential to change the genomic architecture of a genomeat precise locations, with desired accuracy.Several engineered nucleases, including zinc finger nucleases (ZFNs) and TAL effectors nucleases (TALENs) and CRISPR CAS 9 have been used in plants, promising to revolutionize conventional methods of genetic engineering. Targeted editing of the genomes of an organism used to improve productivity and quality of crops.Given the power of genome editing tools and the increasing number of researchers using and developing these tools, a revolutionary change is taking place in crop that resistance to various biotic and abiotic stresses to meet the increasing demand for food and ensure world food security in the future. The reviewhighlights the broad applicability of engineered Nuclease (ZFN, TALEN, and CRISPR CAS 9)mediated targeted plant genome editing and their application for development of designer crops

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Split-TALE: A TALE-based two-component system for synthetic biology applications in planta

Split-TALE: A TALE-based two-component system for synthetic biology applications in planta | TAL effector science | Scoop.it

Schreiber et al, 2019

Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here we design and optimize a two-component AND gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains (IDs). Physical interaction of IDs leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.

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DNA-Free Genome Editing: Past, Present and Future - Frontiers in Plant Science

DNA-Free Genome Editing: Past, Present and Future - Frontiers in Plant Science | TAL effector science | Scoop.it

Metje-Sprink et al, 2019

Genome Editing using engineered endonuclease (GEEN) systems rapidly took over the field of plant science and plant breeding. So far, Genome Editing techniques have been applied in more than fifty different plants; including model species like Arabidopsis; main crops like rice, maize or wheat as well as economically less important crops like strawberry, peanut and cucumber. These techniques have been used for basic research as proof-of-concept or to investigate gene functions in most of its applications. However, several market-oriented traits have been addressed including enhanced agronomic characteristics, improved food and feed quality, increased tolerance to abiotic and biotic stress and herbicide tolerance. These technologies are evolving at a tearing pace and especially the field of CRISPR based Genome Editing is advancing incredibly fast. CRISPR-Systems derived from a multitude of bacterial species are being used for targeted Gene Editing and many modifications have already been applied to the existing CRISPR-Systems such as (i) alter their protospacer adjacent motif (ii) increase their specificity (iii) alter their ability to cut DNA and (iv) fuse them with additional proteins. Besides, the classical transformation system using Agrobacteria tumefaciens or Rhizobium rhizogenes, other transformation technologies have become available and additional methods are on its way to the plant sector. Some of them are utilizing solely proteins or protein-RNA complexes for transformation, making it possible to alter the genome without the use of recombinant DNA. Due to this, it is impossible that foreign DNA is being incorporated into the host genome. In this review we will present the recent developments and techniques in the field of DNA-free Genome Editing, its advantages and pitfalls and give a perspective on technologies which might be available in the future for targeted Genome Editing in plants. Furthermore, we will discuss these techniques in the light of existing– and potential future regulations.

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PrediTALE: A novel model learned from quantitative data allows for new perspectives on TALE targeting - Biorxiv

Erkes et al, 2019, Pre-Print

Plant-pathogenic Xanthomonas bacteria secret transcription activator-like effectors (TALEs) into host cells, where they act as transcriptional activators on plant target genes to support bacterial virulence. TALEs have a unique modular DNA-binding domain composed of tandem repeats. Two amino acids within each tandem repeat, termed repeat-variable diresidues, bind to contiguous nucleotides on the DNA sequence and determine target specificity. In this paper, we propose a novel approach for TALE target prediction to identify potential virulence targets. Our approach accounts for recent findings concerning TALE targeting, including frame-shift binding by repeats of aberrant lengths, and the flexible strand orientation of target boxes relative to the transcription start of the downstream target gene. The computational model can account for dependencies between adjacent RVD positions. Model parameters are learned from the wealth of quantitative data that have been generated over the last years. We benchmark the novel approach, termed PrediTALE, using RNA-seq data after Xanthomonas infection in rice, and find an overall improvement of prediction performance compared with previous approaches. Using PrediTALE, we are able to predict several novel putative virulence targets. However, we also observe that no target genes are predicted by any prediction tool for several TALEs, which we term orphan TALEs for this reason. We postulate that one explanation for orphan TALEs are incomplete gene annotations and, hence, propose to replace promoterome-wide by genome-wide scans for target boxes. We demonstrate that known targets from promoterome-wide scans may be recovered by genome-wide scans, whereas the latter, combined with RNA-seq data, are able to detect putative targets independent of existing gene annotations.

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A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Re...

A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Re... | TAL effector science | Scoop.it

Carpenter et al, 2018

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host ‘susceptibility’ (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harbouring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

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Genome Engineering Using TALENs - in: Barley, Methods and protocols

(via T. Lahaye, thx)

Genome engineering involves methods of genetic modification of cells at predefined genomic sites. Here, we used transcription activator-like effector nucleases (TALENs) for the site-directed mutagenesis in barley. Target gene-specific TALEN-encoding expression units were designed and delivered to totipotent cells of either cultivated embryogenic pollen or immature embryos. The analysis of resulting transgenic plants revealed that the described approach allows for the generation of site-specific, heritable mutations at reasonable efficiency.

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Efficient and orthogonal transcription regulation by chemically inducible artificial transcription factors - Biochemistry

Efficient and orthogonal transcription regulation by chemically inducible artificial transcription factors - Biochemistry | TAL effector science | Scoop.it

Nomura et al, 2018

The DNA-binding specificity of genome editing tools can be applied towards gene regulation. Recently, multiple artificial transcription factors (ATFs) were shown to synergistically and efficiently regulate gene expression. Chemically triggered protein associations are useful for functional regulation at specific timings. A combination of several inducible protein association systems could enable the regulation of multiple genes at different loci with independent timing. We applied the FKBP-rapamycin-FRB and the GAI-Gibberellin-GID systems for gene regulation using multiple TALEs and dCas9. By the combined use of currently available systems, reporter gene assays were performed; the results indicated that gene expression was regulated by rapamycin or gibberellin in the presence of the FRB-, or GAI-effector domains, respectively. Furthermore, the activation of endogenous genes was differentially regulated by the system. This success suggests the usability of the chemically inducible multiple ATFs for the time-dependent regulation of multiple genes, such as the case for cellular phenomena that are dependent on the programmable timing of expression and on the differential expression of multiple genes.

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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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