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.
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.
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.
[...] 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.
[...]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.
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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.
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)
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.
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.
The successful candidate will develop site-specific multigene engineering technology in indica rice using the TAL (transcription activator-like) Effector Nucleases and other approaches at IRRI, applicable to assemble drought tolerance genes and C4 candidate gene,working closely with the collaborators under GRiSP new frontier project. He/She will conduct the site specific integration and multi gene engineering, perform molecular analysis and inheritance study in the transgenic progenies.
Two Blades Foundation (2Blades) has completed a non-exclusive license agreement with KWS SAAT AG (KWS) for access to 2Blades' Transcription Activator Like (TAL) effector code technology for genome engineering in certain crops.
"The TAL Sequence Assembly Tool helps researchers assemble TALE and TALEN plasmids by generating templates and aligning sequencing reads. Compatible with the Golden Gate assembly processes described by Cermak et al. and Sanjana et al. as well as the FLASH assembly process described by Reyon et al."
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."
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.
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.
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.
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.
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.
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
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.”
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.
The DNA-binding domain of transcription activator–like effectors (TALEs) has become an important tool for the programmable and specific targeting of genome-editing nucleases. We determined specificities of TALE DNA-binding modules and discovered varying efficiencies to promote TALE activity.
Specific recognition of guanine is difficult because the common RVD NN (Asn-Asn) recognizes guanine and adenine, whereas the guanine-specific RVD NK (Asn-Lys) apparently functions less well than NN does11. Here we describe new RVD specificities including NH (Asn-His), which is highly specific for guanine. We show that efficiency of RVDs varies and that strong repeats (HD (His-Asn) or NN) are key for overall activity of TALEs.
In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. ...
Mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells.
Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease (TALEN) strategy. We took advantage of the very recently developed “unit assembly” strategy to assemble two pairs of specific TALENs designed to modify the yellow gene (on the sex chromosome) and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected F0 fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.
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