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Scooped by
dromius
September 28, 2012 3:14 AM
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Yin et al (2012) via Tom Schreiber (thanks!) * TALE repeats specifically bind to DNA-RNA hybrids * The sequence of DNA determines binding specificity by TALE repeats * TALE repeats protect DNA-RNA hybrids from RNase H degradation * TALEs may be used to fight retroviral infection and modulate transcription Summary The transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.
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Scooped by
dromius
September 27, 2012 3:06 AM
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(via Tom Schreiber [again! ... keep going]) Valton et al 2012, JBC, Cellectis, France Within the past two years, TAL DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free and universal method to overcome it. Background: TALE-based technologies are poised to revolutionize the field of biotechnology however, their sensitivity to cytosine methylation may drastically restrict the range of their
applications.
Results: TALE repeat N* is able to proficiently bind to 5-methylated cytosine.
Conclusion: Sensitivity of TALE DNA binding domains to cytosine methylation can be overcome by using TALE repeat N*. Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.
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Scooped by
dromius
September 25, 2012 3:25 AM
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Heritable gene targeting in zebrafish using customized TALENs - Nature Biotech.
http://www.nature.com/nbt/journal/v29/n8/full/nbt.1939.html Nature Biotechnology29,699–700(2011) Studies of targeted gene modifications are of great interest in basic research as well as for clinical and agricultural applications1. In the February issue of Nature Biotechnology, two articles reported genomic modifications using transcription activator-like (TAL) effectors2, 3. Using fusion proteins, each comprising a TAL effector DNA binding domain and a FokI cleavage domain, Miller et al.2 reported that TAL effector nucleases (TALENs) successfully disrupted target genes in cultured human cells. Zhang et al.3 showed that TAL effectors can be used to regulate endogenous gene transcription. Compared with zinc-finger proteins4, 5, TAL effectors permit more predictable and specific binding to target DNA6, and therefore allow researchers to engineer genomes precisely without the need for laborious screening to identify a DNA binding domain with the requisite specificity. TALENs can induce DNA double-stranded breaks (DSBs) in yeast7. Gene targeting using TALENs has also been achieved in nematodes8 and human pluripotent cells9. However, it has not, to our knowledge, yet been demonstrated in a vertebrate organism. Here we report the use of TALENs to disrupt both of the two endogenous zebrafish genes we targeted and show that the mutations are transmitted through the germ line.
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Scooped by
dromius
September 24, 2012 7:01 AM
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suggested by Tom Schreiber (thanks!) Establishment of efficient genome editing tools is essential for fundamental research, genetic engineering, and gene therapy. Successful construction and application of transcription activator-like effector nucleases (TALENs) in several organisms herald an exciting new era for genome editing. We describe the production of two active TALENs and their successful application in the targeted mutagenesis of silkworm, Bombyx mori, whose genetic manipulation methods are parallel to those of Drosophila and other insects. We will also show that the simultaneous expression of two pairs of TALENs generates heritable large chromosomal deletion. Our results demonstrate that (i) TALENs can be used in silkworm and (ii) heritable large chromosomal deletions can be induced by two pairs of TALENs in whole organisms. The generation and the high frequency of TALENs-induced targeted mutagenesis in silkworm will promote the genetic modification of silkworm and other insect species.
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Scooped by
dromius
September 16, 2012 10:03 AM
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Stellungnahme der ZKBS zu neuen Techniken für die Pflanzenzüchtung http://www.bvl.bund.de/SharedDocs/Downloads/06_Gentechnik/ZKBS/01_Allgemeine_Stellungnahmen_deutsch/04_Pflanzen/Neue_Techniken_Pflanzenzuechtung.pdf?__blob=publicationFile&v=3 automated translation:
The ZKBS shares the opinion of NTWG that a segment must contain at least 20 nucleotide pairs (NP) to lead to a recombinant nucleic acid. A specific sequence of 20 NP statistically occurs once in a random distribution of 4^20 NP (1.1 x 10^12 NP). Consequently, certain sequences of less than 20 NP are to be expected with a certain probability in large genomes, such as corn (which has 2.5 x 10^9 haploid genome NP). An intentional change of less than 20 NP cannot be distinguished with sufficent confidence from random occurence of this sequence. Certain sequences of less than 20 NP can indeed be detected, however are not suitable for determining their origin. They are indistinguishable from the genetic changes caused by conventional mutagenesis or natural mutation (random occurrence) (Cao et al., 2011). The mutations induced by mutagenesis methods are according to to § 3 no. 3b. Sentence 2 letter a GenTG (mutagenesis) no genetic modifications. Original-Auszug:
"Die ZKBS schließt sich der Meinung der NTWG an, dass ein Segment mindestens 20 Nukleotidpaare (NP) umfassen muss, um zu einer rekombinanten Nukleinsäure zu führen. Eine spezifische Sequenz von 20 NP kommt bei zufälliger Verteilung der NP statistisch einmal in 4^20 NP (1,1 x 10^12 NP) vor. Folglich sind bestimmte Sequenzen von weniger als 20 NP in großen Genomen, wie z. B. Mais (das haploide Genom hat 2,5 x 10^9 NP), mit einer gewissen Wahrscheinlichkeit zu erwarten. Eine absichtliche Änderung von weniger als 20 NP kann von dem zufälligen Vorkommen dieser Sequenz nicht hinreichend sicher unterschieden werden. Bestimmte Sequenzen von weniger als 20 NP können zwar nachgewiesen werden, eignen sich jedoch nicht zur Bestimmung ihrer Herkunft. Sie sind nicht von den durch konventionelle Mutagenese oder natürliche Mutation entstandenen genetischen Veränderungen (zufälliges Vorkommen) zu unterscheiden (Cao et al., 2011). Die durch Mutagenese-Verfahren induzierten Mutationen gelten gemäß § 3 Nr. 3b. Satz 2 Buchst. a GenTG (Mutagenese) nicht als gentechnische Veränderungen."
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Scooped by
dromius
September 14, 2012 7:54 PM
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Recognition of methylated DNA by TAL effectors - Cell Research
Deng et al, 2012 Our experimental characterization provides a molecular basis for distinguishing methylated and unmethylated cytosine. Binding of mC by TALE repeat through the RVD NG extends the DNA recognition code and has potential application in epigenetics and cancer research. For example, specific TALE repeats may be designed to recognize the hypermethylated DNA region; detection can be facilitated by fusing TALEs with fluorescence proteins.
Our study also strongly argues that the in vivo methylation status of the target DNA sequence must be considered for the design of specific DNA-binding TALEs. Methylation of the base C in vivo might render the DNA sequence unfit for binding by the designed TALEs. Because the methylation status of DNA sequences is frequently under dynamic control, one would have to design at least two TALEs for one DNA sequence (i.e., one for methylated and one for unmethylated). In fact, assessment of methylation status of specific DNA sequences in vivo can be greatly facilitated through quantification of fluorescence signal of designed GFP-TALEs. Alternatively, the CpG sequences may be avoided for the application of TALEs, although this practice will somehow limit the potential application.
Despite these complexities, the discovery of mC binding by TALEs with RVD NG opens a number of exciting opportunities.
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Scooped by
dromius
September 7, 2012 5:47 AM
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Many plant-pathogenic bacteria suppress pathogen-associated molecular pattern (PAMP)-triggered immunity by injecting effector proteins into the host cytoplasm during infection through the type III secretion system (TTSS). This type III secretome plays an important role in bacterial pathogenicity in susceptible hosts. Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight, injects several effector proteins into the host cell, including TALE1Xam. This protein is a member of the Transcriptional Activator-Like effector (TALE) protein family, formerly known as the AvrBs3/PthA family. TALE1Xam has 13.5 tandem repeats of 34 amino acids each, as well as two nuclear localization signals and an acidic activation domain at the C-terminus. In this work, we demonstrate the importance of TALE1Xam in the pathogenicity of Xam. We use versions of the gene that lack different domains in the protein in structure–function studies to show that the eukaryotic domains at the 3′ end are critical for pathogenicity. In addition, we demonstrate that, similar to the characterized TALE proteins from other Xanthomonas species, TALE1Xam acts as a transcriptional activator in plant cells. This is the first report [remark: i disagree] of the identification of a TALE in Xam, and contributes to our understanding of the pathogenicity mechanisms employed by this bacterium to colonize and cause disease in cassava.
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Scooped by
dromius
August 28, 2012 4:10 AM
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Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA–protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.
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Scooped by
dromius
August 13, 2012 2:07 AM
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We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications.
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Suggested by
Eli
August 6, 2012 2:19 AM
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Postdoctoral positions are currently available in the Laboratory of Biomolecular Engineering and Nanomedicine, led by Dr. Gang Bao, in the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University. The laboratory is located on the main campus of the Georgia Institute of Technology in Atlanta, GA. The successful candidate will work on genetic engineering strategies involving zinc finger nucleases and TALE nucleases. Tasks include engineering and cloning novel DNA binding domains, testing nuclease function in vitro and in tissue culture. The postdoc will be asked to conduct a range of assays to determine the activity and specificity of the nucleases. The postdoc will also independently design, perform and troubleshoot further experiments to characterize the ability of the nucleases to edit endogenous genes. Candidates must have excellent technical skills, and an enthusiasm for developing and using new techniques.
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Scooped by
dromius
August 5, 2012 4:44 AM
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By taking advantage of solid-phase gene synthesis, we
report herein a novel strategy of magnetic bead-based TALE
assembly, which allowed the synthesis of over one hundred
TALEs, comprised of 16 or 20 repeat units, in three days. We
used a chip containing two components: a microwell array
and magnetic microbeads coated with streptavidin (Figure
1A). Utilizing this chip allowed the simplification of
large-scale TALE production to three steps: monomer
ligation, enzymatic digestion, and purification
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Scooped by
dromius
July 27, 2012 8:45 AM
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One of the sets of tools [Broad Institute core member Feng Zhang and graduate student Le Cong] have developed ... is TAL effectors Scientists have great TAL effector tools for seeking out As, Cs, and Ts, but until now, Gs – which stands for guanine -- have been difficult to target. “For the first time, we were able to demonstrate repression of gene expression by TAL effectors in mammalian cells,” says Le Cong. “This means that now we have bidirectional control – the ability to turn up and turn down gene expression at will.”
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Suggested by
Eli
July 25, 2012 5:06 PM
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Designer DNA-binding proteins based on transcriptional activator-like effectors (TALEs) and zinc finger proteins (ZFPs) are easily tailored to recognize specific DNA sequences in a modular manner. They can be engineered to generate tools for targeted genome perturbation. Here, we review recent advances in these versatile technologies with a focus on designer nucleases for highly precise, efficient, and scarless gene modification. By generating double stranded breaks and stimulating cellular DNA repair pathways, TALE and ZF nucleases have the ability to modify the endogenous genome. We also discuss current applications of designer DNA-binding proteins in synthetic biology and disease modeling, novel effector domains for genetic and epigenetic regulation, and finally perspectives on using customizable DNA-binding proteins for interrogating neural function.
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Scooped by
dromius
September 27, 2012 2:09 PM
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We designed an upgraded bistable genetic toggle switch based on orthogonal TAL repressors and activators which is composed of a pair of mutual repressors and a pair of activators extending the classical toggle swith with positive feedback loops. Simulations of the positive feedback loop switch demonstrated bistability even at low or no cooperativity. Stochastic and deterministic simulations indicate higher robustness in comparison to the mutual repressor switch. We experimentally tested the switch by monitoring production of two fluorescent protein reporters. We confirmed a clear bimodal distribution of reporter fluorescence and demonstrated adoption of stable states by induction with corresponding inducer molecules. The switch persisted in a stable state after the removal of inducer molecules, which confirmed the epigenitc bistability of our system.
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Scooped by
dromius
September 25, 2012 3:26 AM
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In vivo genome editing using a high-efficiency TALEN system - Nature
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11537.html Bedell et al. (2012) Nature The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications1, 2, 3, 4, 5. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns6. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
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Scooped by
dromius
September 25, 2012 3:21 AM
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Christian et al, (2012) suggested Tom Schreiber, thanks! The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths – from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
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Suggested by
Shaun Curtin
September 17, 2012 2:42 PM
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Shaun J. Curtin et al. (2012) Recent advances in the field of genome engineering indicate that it will soon be routine to make site-directed modifications to the genomes of crop species, including targeted mutations, gene insertions, and gene replacements. This new technology will be used to help elucidate gene function and develop new and valuable traits. Key to enabling site-directed genome modifications are sequence-specific nucleases that generate targeted double-stranded DNA breaks in genes of interest. To date, three different sequence-specific nuclease systems have been used in crop plants: zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and LAGLIDADG homing endonucleases, also termed “meganucleases.” In this review, we report on the current state of genome engineering in crop plants, comparing the different nuclease and gene delivery systems. We also consider some of the limitations that nuclease-mediated crop improvement technologies may encounter.
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Scooped by
dromius
September 16, 2012 6:15 AM
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New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases.
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Scooped by
dromius
September 10, 2012 11:11 AM
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Tong et al, 2012 The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.
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Scooped by
dromius
September 5, 2012 4:39 AM
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The Two Blades Foundation (2Blades) and the Monsanto Company announced that they have expanded their TAL Code licensing agreement.
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Scooped by
dromius
August 22, 2012 3:07 AM
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[...] Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.
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Scooped by
dromius
August 8, 2012 4:30 AM
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[...]we incubated cells with fluorescently labeled ZFN or FokI cleavage domain proteins. We observed fluorescence in cell lysate following treatment with ZFN—in the presence or absence of a nuclear localization sequence—but not with the FokI cleavage domain, suggesting that zinc-finger domains facilitate cellular internalization. [...] In direct comparison to Lipofectamine-mediated transient transfection of ZFN expression plasmids, we found that cells subjected to consecutive protein treatments showed a marked decrease in ZFN activity at every off-target site, including the CCR2 locus. Notably, there was no detectable ZFN activity at three of these loci. [...] results indicate that the differences in cleavage specificity could be attributable to the short half-lives of transduced ZFN proteins and that limiting the duration of ZFN exposure inside cells is a viable method for minimizing toxicity. [...] We show that this method can also be used to modify difficult-to-transfect cell types, including patient-derived leukemia cell lines and primary human lymphocytes, supporting the use of this technique in place of viral-mediated gene delivery for inducing gene knockouts in cultured cells for reverse genetics and drug discovery. As methods for engineering cell permeability into proteins improve, we anticipate that protein delivery and the benefits afforded therein will be extended to other designer nucleases, including TALENs.
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Scooped by
dromius
August 5, 2012 5:02 AM
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Unfortunately there is a typo in this manuscript - Xa17 does not exist , the authors most likely refer to Xa7 (see below) Increasing evidence indicates that phytopathogens have adopted a number of strategies to coopt plant physiological processes to sustain microbial growth and proliferation. In rice, recessive alleles of the xa13 gene confer resistance to Xanthomonas oryzae pv. oryzae (Xoo). In rice cultivars carrying dominant Xa13 alleles, the bacterial transcription activator-like (TAL) effector PthXoI binds to the Xa13 promoter, leading to increased expression in leaf vascular tissues and facilitating the onset of bacterial blight (Yang et al., 2006). Resistance occurs when mutations in the xa13 promoter prevent PthXoI binding (Chu et al., 2006). A similar mechanism was found for the xa17 resistance gene and its corresponding TAL effector AvrXa7 (Antony et al., 2010)
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Scooped by
dromius
July 27, 2012 8:47 AM
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How TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.
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Scooped by
dromius
July 27, 2012 8:40 AM
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Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains - Nature Communications
http://www.nature.com/ncomms/journal/v3/n7/full/ncomms1962.html Transcription activator-like effectors are sequence-specific DNA-binding proteins that harbour modular, repetitive DNA-binding domains. Transcription activator-like effectors have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the transcription activator-like effector toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat-variable diresidue Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective transcription activator-like effector transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using transcription activator-like effectors.
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