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Rescooped by Christian Faltado Cantos from plant cell genetics
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Visit to vandalized Golden Rice field trial - IRRI (2013)

IRRI and PhilRice officials visit the Golden Rice field site that was vandalized... A crowd of 300 had stormed the Department of Agriculture (DA) Regional Field Unit 5's (RFU5) Bicol Experiment Station, overwhelming the police and guards, and vandalizing the research plots of Golden Rice... 

 

Regional Executive Diretor Bragas said that they were taken by surprise. They had assembled DA officials and staff in the office, waiting for the group to come in and sit down for a peaceful dialogue. Instead, the militants poured into the compound, overwhelmed the police and village security, broke down a section of the fence surrounding the research area, and entered, uprooted, and trampled the crop. 

 

The officials shared that there were farmers in the group, but they just watched and stayed on the sidelines. Local customs and traditions dictate that the destruction of a living field brings bad fortune – Bicolanos refer to it as "Bosung". Those who entered the field to vandalize were mostly young men and some covered their faces. 

 

The local officials and the Institutional Biosafety Committee (IBC) recounted that the rallyists had been assembled in Legazpi City the day before, and brought overnight to Naga City in a convoy of about a dozen jeepneys. In Naga City they had been housed in local hotels. It was reported that the group included foreigners. 


Via Alexander J. Stein, Jean-Pierre Zryd
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Alexander J. Stein's curator insight, August 11, 2013 10:57 PM

Interesting to read how little regard the anti-GMO activists apparently had for local customs, traditions and beliefs (which otherwise they probably claim to be dear to their heart), simply overpowering local farmers and stakeholders rather than empowering them. (A jeepney can easily accommodate more than a dozen passengers, i.e. a dozen jeepneys can have bussed in more than half of the "farmers" from Legazpi City who vandalised the field trial.) 

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Plant Cell: Plant Immune Responses Against Viruses: How Does a Virus Cause Disease?

Plant Cell: Plant Immune Responses Against Viruses: How Does a Virus Cause Disease? | Multi- gene | Scoop.it

New review article in Plant Cell.

"Recently, significant progress has been made in understanding RNA silencing and how viruses counter this apparently ubiquitous antiviral defense. In addition, plants also induce hypersensitive and systemic acquired resistance responses, which together limit the virus to infected cells and impart resistance to the noninfected tissues."


Via Mary Williams, Ricardo Oliva
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Andres Zurita's curator insight, May 29, 2013 8:28 AM

Open Access pdf

María Serrano's curator insight, June 24, 2014 12:30 PM
Respuesta inmune de las plantas frente a los virus.
Rescooped by Christian Faltado Cantos from Amazing Science
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Editing the genome with high precision and inserting multiple genes into specific locations

Editing the genome with high precision and inserting multiple genes into specific locations | Multi- gene | Scoop.it

Researchers at MIT, the Broad Institute and Rockefeller University have developed a new technique for precisely altering the genomes of living cells by adding or deleting genes. The researchers say the technology could offer an easy-to-use, less-expensive way to engineer organisms that produce biofuels; to design animal models to study human disease; and  to develop new therapies, among other potential applications.

To create their new genome-editing technique, the researchers modified a set of bacterial proteins that normally defend against viral invaders. Using this system, scientists can alter several genome sites simultaneously and can achieve much greater control over where new genes are inserted, says Feng Zhang, an assistant professor of brain and cognitive sciences at MIT and leader of the research team.  

“Anything that requires engineering of an organism to put in new genes or to modify what’s in the genome will be able to benefit from this,” says Zhang, who is a core member of the Broad Institute and MIT’s McGovern Institute for Brain Research.

 

Complexes known as transcription activator-like effector nucleases (TALENs) can also cut the genome in specific locations, but these complexes can also be expensive and difficult to assemble. The new system is much more user-friendly than TALENs, Zhang says. Making use of naturally occurring bacterial protein-RNA systems that recognize and snip viral DNA, the researchers can create DNA-editing complexes that include a nuclease called Cas9 bound to short RNA sequences. These sequences are designed to target specific locations in the genome; when they encounter a match, Cas9 cuts the DNA. 

This approach can be used either to disrupt the function of a gene or to replace it with a new one. To replace the gene, the researchers must also add a DNA template for the new gene, which would be copied into the genome after the DNA is cut. 

Each of the RNA segments can target a different sequence. “That’s the beauty of this — you can easily program a nuclease to target one or more positions in the genome,” Zhang says. 

The method is also very precise — if there is a single base-pair difference between the RNA targeting sequence and the genome sequence, Cas9 is not activated. This is not the case for zinc fingers or TALEN. The new system also appears to be more efficient than TALEN, and much less expensive. 


Via Dr. Stefan Gruenwald
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RNA-guided editing of bacterial genomes using CRISPR-Cas systems

RNA-guided editing of bacterial genomes using CRISPR-Cas systems | Multi- gene | Scoop.it
A CRISPR-Cas system is harnessed to introduce template-driven mutations in S. pneumoniae and E. coli at high efficiency without requiring selectable markers.

Via Gerd Moe-Behrens
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Gerd Moe-Behrens's curator insight, January 30, 2013 5:49 AM

Congratulation +David Bikard 

*RNA-guided editing of bacterial genomes using CRISPR-Cas systems*

by
Wenyan Jiang,David Bikard,David Cox,Feng Zhang& Luciano A Marraffini

"Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering."

http://bit.ly/11dWGDK

see also http://bit.ly/VvkdOY

Rescooped by Christian Faltado Cantos from Amazing Science
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Gene Therapy's Next Frontier: Zinc-Finger Nucleases for Somatic Gene Therapy

Gene Therapy's Next Frontier: Zinc-Finger Nucleases for Somatic Gene Therapy | Multi- gene | Scoop.it

Zinc-finger nucleases (ZFNs) are a powerful tool that can be used to edit the human genome ad libitum. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to generate zinc-finger proteins. As a result, two phase I clinical trials aimed at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these lymphocytes from infection with the virus have been initiated. Moreover, ZFNs have been successfully employed to knockout or correct disease-related genes in human stem cells, including hematopoietic precursor cells and induced pluripotent stem cells. Targeted genome engineering approaches in multipotent and pluripotent stem cells hold great promise for future strategies geared toward correcting inborn mutations for personalized cell replacement therapies.

 

Short-lived ZFN expression from episomal DNA-based expression vectors—such as plasmid DNA, integrase-deficient lentiviral vectors, adenoviral vectors, and vectors based on adeno-associated virus—can only be achieved in mitotic cells, which ensures rapid dilution of the vectors during cell divisions. Because DNA-based vector systems have a tendency to integrate into the host genome, it will be important to closely follow the fate of the ZFN expression vectors in the target cells. An alternative way of delivering ZFNs is the transfer of ZFN-encoding mRNA, which ensures rapid but transient ZFN expression and avoids the issue of illegitimate integration.

 

Microinjection of ZFN-encoding mRNA has been performed in zebrafish and rat single-cell embryos, and the ZFN-mediated gene disruption frequency was comparable to plasmid DNA delivery. Moreover, delivery of ZFNs by mRNA transfection has been used to target the integration of a transgene into the AAVS1 locus in human iPSCs.

 

If direct in situ correction of a disease locus is not an option, an important consideration will be to determine where to integrate a therapeutic transgene cassette into the human genome. The AAVS1 site on chromosome 19 is thus far the most promising candidate for such a safe harbor, as a native insulator region appears to both protect transgene expression from position-effect variegation and silencing and prevent the transgene promoter from affecting the host transcriptome.

 

The fact that ZFNs can be used to create knockout animals is especially encouraging and emphasizes the high specificity the technology has reached in the last 3 years. Moreover, the development of alternative designer nucleases, such as TALENs and meganucleases, has further spurred interest in targeted genome engineering approaches. Conversely, studies reporting ZFN off-target activities in zebrafish and human cells must not be overlooked and should serve as the basis for further improvement of the technology. The employment of highly specific designer nucleases is especially important when DSB-based genome engineering is applied to multipotent or pluripotent stem cells, such as HSCs or iPSCs, with their high proliferative potential. Even so, the remarkable progress achieved in the last few years demonstrates that ZFNs represent a tool that allows researchers and clinicians for the first time to rationally edit the genome of human cells and to take this technology from the bench to the bedside for therapeutic applications.


Via Dr. Stefan Gruenwald
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Forbes: CRISPR-associated nuclease could change biotech forever

Forbes: CRISPR-associated nuclease could change biotech forever | Multi- gene | Scoop.it

"A tiny molecular machine used by bacteria to kill attacking viruses could change the way that scientists edit the DNA of plants, animals and fungi, revolutionizing genetic engineering."


Via Mary Williams
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CRISPR/Cas System Genome Editing - EpiGenie

CRISPR/Cas System Genome Editing - EpiGenie | Multi- gene | Scoop.it
CRISPR/Cas is getting a lot of attention lately as a way to insert or delete genomic sequences in mammalian cells.
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Cell - One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering http://t.co/d5E3AmtrfK via Cell
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Biotechnology: Rewriting a genome | CRISPR-Cas ...

Biotechnology: Rewriting a genome | CRISPR-Cas ... | Multi- gene | Scoop.it
A bacterial enzyme that uses guide RNA molecules to target DNA for cleavage has been adopted as a programmable tool to site-specifically modify genomes of cells and organisms, from bacteria and human cells to whole zebrafish.
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TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants - Plant Mol. Biol.

TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants - Plant Mol. Biol. | Multi- gene | Scoop.it

(via T. Lahaye, thx)

Wendt et al, (2013)

Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.


Via dromius
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Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease - New Phytologist

(Via T. Lahaye & T. Schreiber) De Lange et al 2013 Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5′ terminal guanine, contrasting with the TALE preference for a 5′ thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.


Via dromius
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PLOS ONE: Comparative Genomic and Transcriptomic Analysis of Tandemly and Segmentally Duplicated Genes in Rice

PLOS ONE: Comparative Genomic and Transcriptomic Analysis of Tandemly and Segmentally Duplicated Genes in Rice | Multi- gene | Scoop.it

Tandem and segmental duplications significantly contribute to gene family expansion and genome evolution. Genome-wide identification of tandem and segmental genes has been analyzed before in several plant genomes. However, comparative studies in functional bias, expression divergence and their roles in species domestication are still lacking. We have carried out a genome-wide identification and comparative analysis of tandem and segmental genes in the rice genome. A total of 3,646 and 3,633 pairs of tandem and segmental genes, respectively, were identified in the genome. They made up around 30% of total annotated rice genes (excluding transposon-coding genes). Both tandem and segmental duplicates showed different physical locations and exhibited a biased subset of functions. These two types of duplicated genes were also under different functional constrains as shown by nonsynonymous substitutions per site (Ka) and synonymous substitutions per site (Ks) analysis. They are also differently regulated depending on the tissues and abiotic and biotic stresses based on transcriptomics data. The expression divergence might be related to promoter differentiation and DNA methylation status after tandem or segmental duplications. Both tandem and segmental duplications differ in their contribution to genetic novelty but evidence suggests that they play their role in species domestication and genome evolution


Via Dorian Q Fuller, Ricardo Oliva
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Dorian Q Fuller's curator insight, May 19, 2013 3:25 PM

Shows there are quite alot of duplicated genes in the rice genome. Unfortunately, the number that were involved in the domestication process remains speculative-- and needs to be investigated!

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ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering - Trends in Biotechnol.

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering - Trends in Biotechnol. | Multi- gene | Scoop.it

(via T. Lahaye, thx)

Gaj et al, 2013

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.


Via dromius
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Clem Stanyon's curator insight, May 13, 2013 12:16 AM

The efficiencies are getting into the double-digit percentages, these days, though that was first achieved over 10 years ago with meganucleases curring the genome at defined sites. These systems will certainly be part of the future genome-editing suite, but have some distance to go before in situ genome engineering is efficient enough to be routine.

 

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Targeted variation of genomes using TAL effectors - plantsynbio workshop Nottingham

Slides from my presentation in Nottingham 2013


Via dromius
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The Planetary Archives / San Francisco, California's curator insight, June 6, 2013 3:17 PM

These people need to be stopped before they create something they can't control, if they haven't already.....

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A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta - Plant Mol. Biol.

A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta - Plant Mol. Biol. | Multi- gene | Scoop.it

(via T. Lahaye, thanks...cant beat his speed :))

Johnson et al, 2013

Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase® reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases’ cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established ‘QQR’ zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.


Via dromius
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Genome Engineering of Crops with Designer Nucleases

Genome Engineering of Crops with Designer Nucleases | Multi- gene | Scoop.it

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.

 

More TALEN news on: http://www.scoop.it/t/tal-effector-science


Via dromius, Dr. Stefan Gruenwald
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Multiplex Genome Engineering Using CRISPR/Cas Systems

Multiplex Genome Engineering Using CRISPR/Cas Systems | Multi- gene | Scoop.it

Via Gerd Moe-Behrens
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Gerd Moe-Behrens's curator insight, February 15, 2013 9:04 AM

by
Le Cong, F. Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano A. Marraffini, Feng Zhang

"Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology."

 http://bit.ly/VVv3yb

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A CRISPR way to engineer the human genome - Genome Biology

Research highlight. A CRISPR way to engineer the human genome. Sivaprakash Ramalingam , Narayana Annaluru and Srinivasan Chandrasegaran. Genome Biology 2013, 14:107 (26 February 2013). Abstract | Full text | PDF | PubMed ...
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“Genomic Cruise Missiles” (TALENS) Used to Alter Mosquito ...

“Genomic Cruise Missiles” (TALENS) Used to Alter Mosquito Genome. Fri, 03/22/2013 - 08:19 — bioquicknews. In a study recently published online on March 21, 2013 in the open-access journal PLOS ONE, Virginia Tech scientists used a pair ...
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Double-strand DNA end-binding and sliding of the toroidal CRISPR ...

Double-strand DNA end-binding and sliding of the toroidal CRISPR ... | Multi- gene | Scoop.it
Abstract. The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading ...
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No Punnett Tended: TALENs -- Huh? - blog*spot

No Punnett Tended: TALENs -- Huh? - blog*spot | Multi- gene | Scoop.it
The newly-developed transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be easily engineered so that TALENs can target ...
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CRISPR Based System for Targeted Reduction of Gene Expression ...

CRISPR Based System for Targeted Reduction of Gene Expression ... | Multi- gene | Scoop.it
Application A CRISPR based gene silencing system using sequence specific RNA to repress expression of endogenous transcripts. Key Benefits Provides a platform to target specific mRNA transcripts i...
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Addgene: Addgene's CRISPR Guide

Addgene: Addgene's CRISPR Guide | Multi- gene | Scoop.it
RT @plantscience @weedinggems: Beyond TALE: CRISPR/CAS9 nucleases http://t.co/CVkVOG4dWB #plantsynbio
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Generation of gene disruptions by transcription activator-like effector ...

Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos - up-to-the-minute news and headlines. 7thSpace is a online portal covering topics such as Family, ...
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Science names U gene-modification technique a 2012 top breakthrough - Minnesota Medical Foundation (blog)

Science names U gene-modification technique a 2012 top breakthrough - Minnesota Medical Foundation (blog) | Multi- gene | Scoop.it
Minnesota Medical Foundation (blog) Science names U gene-modification technique a 2012 top breakthrough Minnesota Medical Foundation (blog) With their high level of accuracy, TALENs hold the promise of correcting genetic diseases without the risks...
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