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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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Developing World Cities and Population Density

Developing World Cities and Population Density | Multi- gene | Scoop.it
Without a question, we are living in an urban era. More people now live in cities than anywhere else on the planet and I’ve repeatedly argued that cities are our most important economic engine. As a result of these shifts, we’re seeing megacities at a scale the world has never seen before.

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Mr Steven Newman's curator insight, June 26, 2:25 PM

Just a few years ago there were only  20 cities with a pop over 10 million.  I'll use this with my  10 geography class to compare Australia's population and size with these cities and  look at debates around policies.  I,ll try and get the students to debate around ecological sustainability.

Fathie Kundie's curator insight, June 27, 9:05 AM
المدن الأعلى كثافة بالسكان على مستوى العالم
Sally Egan's curator insight, June 29, 6:31 PM

Mega cities and the challenges they face for the future is focus in this article. Great statistics on populations and urban densities are also included.

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Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations

Efficient CRISPR/Cas9-Mediated Gene Editing in Arabidopsis thaliana and Inheritance of Modified Genes in the T2 and T3 Generations | Multi- gene | Scoop.it
PLOS ONE: an inclusive, peer-reviewed, open-access resource from the PUBLIC LIBRARY OF SCIENCE. Reports of well-performed scientific studies from all disciplines freely available to the whole world.
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Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops

Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops | Multi- gene | Scoop.it

Scooped from: Molecular Plant, 2013

Authors: Kabin Xie, Jianwei Zhang and Yinong Yang

 

Genome-wide prediction of highly specific guide RNA (gRNA) spacer sequences  in eight model plants and agricultural crop species, including 

Arabidopsis thaliana, Medicago truncatula, Glycine max (soybean), Solanum lycopersicum (tomato), Brachypodium distachyon, Oryza sativa (rice), Sorghum bicolor and Zea mays (maize).

 

CRISPR-PLANT Database - http://www.genome.arizona.edu/crispr "enables the plant research community to access genome-wide predictions of specific gRNAs, and facilitate the application of CRISPR-Cas9 mediated genome editing in model plants and major agricultural crops".

 


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Plant Cell: Geminivirus Vectors Deliver Reagents for Plant Genome Engineering (2014)

Plant Cell: Geminivirus Vectors Deliver Reagents for Plant Genome Engineering (2014) | Multi- gene | Scoop.it

Jennifer Mach commentary http://www.plantcell.org/content/early/2014/01/16/tpc.114.122606.full.pdf+html

 

Baltes et al. article http://www.plantcell.org/content/early/2014/01/16/tpc.113.119792.abstract


Via Kamoun Lab @ TSL, Ricardo Oliva
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Novel Genetic Patterns May Make Us Rethink Biology and Individuality - The Almagest

Novel Genetic Patterns May Make Us Rethink Biology and Individuality - The Almagest | Multi- gene | Scoop.it

Professor of Genetics Scott Williams, PhD, of the Institute for Quantitative Biomedical Sciences (iQBS) at Dartmouth’s Geisel School of Medicine, has made two novel discoveries: first, a person can have several DNA mutations in parts of their body, with their original DNA in the rest—resulting in several different genotypes in one individual—and second, some of the same genetic mutations occur in unrelated people. We think of each person’s DNA as unique, so if an individual can have more than one genotype, this may alter our very concept of what it means to be a human, and impact how we think about using forensic or criminal DNA analysis, paternity testing, prenatal testing, or genetic screening for breast cancer risk, for example. Williams’ surprising results indicate that genetic mutations do not always happen purely at random, as scientists have previously thought. His work, done in collaboration with Professor of Genetics Jason Moore, PhD, and colleagues at Vanderbilt University, was published in PLOS Genetics journal.[1]


Genetic mutations can occur in the cells that are passed on from parent to child and may cause birth defects. Other genetic mutations occur after an egg is fertilized, throughout childhood or adult life, after people are exposed to sunlight, radiation, carcinogenic chemicals, viruses, or other items that can damage DNA. These later or “somatic” mutations do not affect sperm or egg cells, so they are not inherited from parents or passed down to children. Somatic mutations can cause cancer or other diseases, but do not always do so. However, if the mutated cell continues to divide, the person can develop tissue, or a part thereof, with a different DNA sequence from the rest of his or her body.

 

“We are in reality diverse beings in that a single person is genetically not a single entity—to be philosophical in ways I do not yet understand—what does it mean to be a person if we are variable within?” says Williams, the study’s senior author, and founding Director of the Center for Integrative Biomedical Sciences in iQBS. “What makes you a person? Is it your memory? Your genes?” He continues, “We have always thought, ‘your genome is your genome.’ The data suggest that it is not completely true.”

 

In the past, it was always thought that each person contains only one DNA sequence (genetic constitution). Only recently, with the computational power of advanced genetic analysis tools that examine all the genes in one individual, have scientists been able to systematically look for this somatic variation. “This study is an example of the type of biomedical research project that is made possible by bringing together interdisciplinary teams of scientists with expertise in the biological, computational and statistical sciences.” says Jason Moore, Director of the iQBS, who is also Associate Director for Bioinformatics at the Cancer Center, Third Century Professor, and Professor of Community and Family Medicine at Geisel.

 

Having multiple genotypes from mutations within one’s own body is somewhat analogous to chimerism, a condition in which one person has cells inside his or her body that originated from another person (i.e., following an organ or blood donation; or sometimes a mother and child—or twins—exchange DNA during pregnancy. Also, occasionally a person finds out that, prior to birth, he or she had a twin who did not survive, whose genetic material is still contained within their own body).[2] Chimerism has resulted in some famous DNA cases: one in which a mother had genetic testing that “proved” that she was unrelated to two of her three biological sons.[3]


As suggested by Maria Schnee (newphoenix.info)


1 Williams, Scott, et al., Recurrent tissue-specific mtDNA mutations are common in humans. http://www.plosgenetics.org/doi/pgen.1003929.


2 Strain L, Dean JC, Hamilton MP, Bonthron D. A true hermaphrodite chimera resulting from embryo amalgamation after in vitro fertilization. N Engl J Med 1998;(338):166-9/


3 Norton AT and Zehner O. Project MUSE: Today’s Research, Tomorrow’s Inspiration. http://www.academia.edu.

 
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Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors - PNAS

Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors - PNAS | Multi- gene | Scoop.it

(via T. Lahaye, thx)

Ma et al, 2013

We describe a transcription activator-like effector (TALE)-based strategy, termed “TALEColor,” for labeling specific repetitive DNA sequences in human chromosomes. We designed TALEs for the human telomeric repeat and fused them with any of numerous fluorescent proteins (FPs). Expression of these TALE–telomere–FP fusion proteins in human osteosarcoma's (U2OS) cells resulted in bright signals coincident with telomeres. We also designed TALEs for centromeric sequences unique to certain chromosomes, enabling us to localize specific human chromosomes in live cells. Meanwhile we generated TALE–FPs in vitro and used them as probes to detect telomeres in fixed cells. Using human cells with different average telomere lengths, we found that the TALEColor signals correlated positively with telomere length. In addition, suspension cells were followed by imaging flow cytometry to resolve cell populations with differing telomere lengths. These methods may have significant potential both for basic chromosome and genome research as well as in clinical applications.

 


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Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System - J. Gen. Genomics

Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System - J. Gen. Genomics | Multi- gene | Scoop.it

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Liang et al, 2013

Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition the CRISPR/Cas system induced targeted mutations in Zea mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.


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Ilyas Muhammad's comment, December 17, 2013 3:19 AM
thnx, Seb
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An efficient strategy for TALEN-mediated genome engineering in Drosophila

An efficient strategy for TALEN-mediated genome engineering in Drosophila | Multi- gene | Scoop.it

(via T. Lahaye)

Katsuyama et al, 2013

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.


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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. 


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Alexander J. Stein's curator insight, August 11, 2013 7:57 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."


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Andres Zurita's curator insight, May 29, 2013 5:28 AM

Open Access pdf

María Serrano's curator insight, June 24, 9:30 AM
Respuesta inmune de las plantas frente a los virus.
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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. 


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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.

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Socrates Logos's curator insight, January 30, 2013 2:49 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

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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.


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Frontiers | Identification of “safe harbor” loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair | Plant Physiology

Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specifi...
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How to live a GMO-free life: Hear the 'Awakenings' podcast from the Health Ranger

How to live a GMO-free life: Hear the 'Awakenings' podcast from the Health Ranger | Multi- gene | Scoop.it
How to live a GMO-free life: Hear the 'Awakenings' podcast from the Health Ranger

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Approvals of GM crops in the European Union - January 2014 update

Approvals of GM crops in the European Union - January 2014 update | Multi- gene | Scoop.it
Information Services for Seed Professionals - The Best Place on the Web for Seed Professionals

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Genome Editing with CRISPR Systems - Genetic Engineering News

Genome Editing with CRISPR Systems - Genetic Engineering News | Multi- gene | Scoop.it
Genome Editing with CRISPR Systems Genetic Engineering News CRISPR endonucleases have been used in a recent flood of literature due to their elegant and simple mode of RNA-guided gene targeting and ability to operate via protocols developed...
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Targeting and tracing of specific DNA sequences with dTALEs in living cells - Nucl. Acids Res.

Targeting and tracing of specific DNA sequences with dTALEs in living cells - Nucl. Acids Res. | Multi- gene | Scoop.it

(via T. Lahaye, thx)

Thanisch et al, 2013

Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.


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TALE Activators Regulate Gene Expression in a Position- and Strand-dependent Manner in Mammalian Cells - Biochem. Biophys. Res. Comm.

TALE Activators Regulate Gene Expression in a Position- and Strand-dependent Manner in Mammalian Cells - Biochem. Biophys. Res. Comm. | Multi- gene | Scoop.it

(via T. Lahaye, thx)

Uhde-Stone et al, 2013

Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.


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Six easy steps to avoid common genetically modified foods

Six easy steps to avoid common genetically modified foods | Multi- gene | Scoop.it
Six easy steps to avoid common genetically modified foods

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Skip Stein's curator insight, August 11, 2013 4:55 AM

And the very BEST way is to 'scan it before you buy it' with FOODUCATE.  It will tell you all the ingredients in a processed/packaged food (if it's not in the data base, you can send in the nutrition ifo!).  Its a FREE app for your android or iphone! Join the millions of US who are checking and reading the labels with FOODUCATE! http://www.fooducate.com/

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Genome Modification—a Practical Approach | AlleleBlog

AlleleBlog entry on #chromosome_modification http://t.co/0A6sjmPLpN referenced in GEN article by Patricia Dimond http://t.co/jrEhYU7LLe
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Targeted variation of genomes using TAL effectors - plantsynbio workshop Nottingham

Slides from my presentation in Nottingham 2013


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San Francisco Times-Star™ 's curator insight, June 6, 2013 12:17 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.


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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


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Multiplex Genome Engineering Using CRISPR/Cas Systems

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

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Socrates Logos's curator insight, February 15, 2013 6:04 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."

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